Mutated penicillin G acylase genes

ABSTRACT

New mutant  beta -lactam Penicillin G acylases are provided, exhibiting altered substrate specificities. These Penicillin G acylases are obtained by expression of a gene encoding said Penicillin G acylase and having an amino acid sequence which differs at least in one amino acid from the wild-type Penicillin G acylase.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 08/793,229, filedApr. 23, 1997 now issued as U.S. Pat. No. 5,891,703, which itself is anational phase application of PCT International Application No. 96/05318filed Aug. 14, 1995 as PCT/EP95/03249. Additionally, this applicationclaims the priority of European Patent Application No. EP 942023144filed in the Netherlands on Aug. 12, 1994.

FIELD OF THE INVENTION

The present invention relates to mutations of prokaryotic Penicillin Gacylase or its preenzyme or preproenzyme, resulting in alteredproperties of the mutant penicillin G acylase.

BACKGROUND OF THE INVENTION

The basic antibiotics of the β-lactam type are principally obtained byfermentation. Fungi of the genus Penicillium and Cephalosporium(Acremonium) are used for the production of raw material for β-lactamantibiotics as penicillin G, penicillin V and cephalosporin C. Thesefermentation products, also referred to as PenG, PenV and CefC,respectively, are the starting materials for nearly all currentlymarketed penicillins and cephalosporins. In general the acyl group atthe 6-amino of the penicillin nucleus or at the 7-amino position of thecephalosporin nucleus is referred to as `side chain`, the correspondingacid as `side chain acid`. The side chains of PenG, PenV and CefC arephenylacetyl, phenoxy-acetyl and aminoadipyl, respectively. The sidechains are removed by cleavage of an amide linkage (deacylation),resulting in 6-aminopenicillanic acid (6-APA) in case of the penicillinmolecules and 7-aminocephalosporanic acid (7-ACA) in case of thecephalosporin molecule. In this respect alsophenylacetyl-7-aminodesacetoxycepha-losporanic acid (CefG) should bementioned as a precursor of 7-ADCA, although CefG is not a fermentationproduct. CefG is usually produced chemically from Penicillin G.

In order to obtain β-lactam compounds with an altered activity spectrum,an increased resistance against β-lactanases or improved clinicalperformance of β-lactam compounds, 6-APA, 7-ACA and 7-ADCA are used asstarting points for synthetic manipulation to produce the variouspenicillins and cephalosporins of choice. At present these semisyntheticpenicillins and cephalosporins form by far the most important market ofβ-lactam antibiotics.

The production of semisynthetic β-lactam products requires thedeacylation of the penicillins and cephalosporins produced fromfermentation. Although rather efficient chemical routes are availablefor the deacylation (J. Verweij & E. de Vroom, Recl. Trav. Chim.Pays-Bas 112 (1993) 66-81), nowadays the enzymatic route is preferred inview of the high energy and solvents cost together with someenvironmental problems associated with the chemical route (Dunnill, P.,Immobilised Cell and Enzyme Technology. Philos, Trans. R. Soc. LondonB290 (1980) 409-420). The enzymes which may accomplish the deacylationof β-lactam compounds are classified as hydrolases based on the chemicalreaction they catalyse. However, those hydrolases which are particularlyuseful in the deacylation is of β-lactam compounds are usually referredto in the art as `acylases` or `amidases`. These denominations as usedin this specification have the same meaning. In connection with β-lactamantibiotics these acylases usually are further specified as `β-lactamacylases` as not all amidases accept a β-lactam nucleus as anacceptor/donor moiety for the acyl group. According to the literatureseveral types of β-lactam acylases may be envisaged, based on theirsubstrate specificity and molecular structure (B. S. Deshpande et al.,World J. Microbiology & Biotechnology 10 (1994) 129-138).

Acylase, Nomenclature & Classification

Classification according to Specificity.

The substrate specificity of the acylase is determined by a side chainbinding pocket at the enzyme which recognizes the side chain moiety ofβ-lactam molecules. In general, the acylases are not very specific forthe moiety adjacent to the nitrogen atom of the amide group (this mightbe a cephem group, a penem group, an amino acid, sugars, etc. (J. G.Shewale et al., Process Biochemistry International, Jun. 1990, 97-103).In case of the Penicillin G acylases (Benzylpenicillin amidohydrolase,also named Penicillin amidase; EC 3.5.1.11) this acyl moiety must bevery hydrophobic and is preferably phenylacetyl or (short) alkyl.Penicillin G acylase is used commercially to hydrolyse PenG or CefG tophenylacetic-acid and 6-APA or 7-ADCA, respectively the most importantintermediates for the industrial production of semi-syntheticpenicillins and cephalosporins. Beside these major applications otherhave been reported for these enzymes such as blocking/deblocking ofsensitive groups in organic synthesis and peptide chemistry,stereospecific conversions, optical resolution of phenylglycine,deesterification of carbinols, acylation of mono-bactams etc. In thevarious applications the enzyme may be used C. either in its nativestate or as immobilised preparation. Microbial whole cells containingthe enzyme activity have also been used either as cell suspension or asimmobilised cell preparation.

Examples of substrates which are not hydrolyzed by Penicillin G acylasesare those with charged acyl moieties such as dicarboxylic acids:succinyl, glutaryl, adipyl and also amino-adipyl, the side-chain ofCefC.

Penicillin V acylases are highly specific for phenoxyacetyl, whileampicillin acylase prefers D-phenylglycine as a side chain.Glutaryl-acylases deacylate glutaryl-7-ACA, which is prepared from CefCafter enzymatic deamidation of the side chain with D-amino acid oxidasefollowed by chemical decarboxylation of the formed ketoadipyl derivativewith peroxide, which is produced in the first step. Moreover some ofthese acylases have been reported to be capable of hydrolyzingcephalosporins (including the desacetoxy-derivative) with succinyl,glutaryl and adipyl as an acyl moiety and even in one case CefC to avery limited degree (for a review see EP-A-322032, Merck). So far theseacylases have only been found in Pseudomonas species, and in certainstrains of Bacillus megaterium and Arthrobacter viscosus.

Classification based on structural properties of the enzymes.

Apart from their specificities acylases may also be classified based onmolecular aspects (V. K. sudhakaran et al., Process Biochemistry 27(1992) 131-143):

Type-I acylases are specific for Penicillin V. These enzymes arecomposed of four identical subunits, each having a molecular weight of35 kDa.

Type-II acylases all share a common molecular structure: these enzymesare heterodimers composed of a small subunit (α; 16-26 kDa) and a largesubunit (β; 54-66 kDa). With respect to the substrate specificity,Type-II acylases may be further divided into two groups:

Type-IIA acylases comprise the Penicillin G acylases;

Type-IIB acylases comprise the Glutaryl acylases.

Type III acylases are the Ampicillin acylases which have been reportedto be dimers consisting of two identical subunits with a molecularweight of 72 kDa.

Benefits of Protein Encineerina with Respect to Screening/ChemicalModification

Enzymes with improved properties can be developed or found in severalways, for example, by classical screening methods, by chemicalmodification of existing proteins, or by using modern genetic andprotein-engineering techniques.

Screening for organisms or microorganisms that display the desiredenzymatic activity, can be performed, for example, by isolating andpurifying the enzyme from a microorganism or from a culture supernatantof such microorganisms, determining its biochemical properties andchecking whether these biochemical properties meet the demands forapplication. The present collection of acylases results from intensivescreening programs. β-lactam acylase activity has been found in manymicroorganisms such as fungi, yeast, actinomycetes and bacteria.

If the identified enzyme cannot be obtained from its natural producingorganism, recombinant-DNA techniques may be used to isolate the geneencoding the enzyme, express the gene in another organism, isolate andpurify the expressed enzyme and test whether it is suitable for theintended application.

Modification of existing enzymes can be achieved inter alia by chemicalmodification methods. In general, these methods are too unspecific inthat they modify all accessible residues with common side chains or theyare dependent on the presence of suitable amino acids to be modified,and often they are unable to modify amino acids difficult to reach,unless the enzyme molecule is unfolded. In addition chemicalmodification require Additional processing steps and chemicals toprepare the enzyme. Enzyme modification through mutagenesis of theencoding gene does not suffer from the problems mentioned above, andtherefore is thought to be superior.

Moreover the choice for an acylase, subsequent construction andselection of high-yielding penicillin acylase-producing strains and thedevelopment of an industrial process for isolation and immobilisation,is a laborious process. In general for production and subsequentformulation of the mutants the wild type protocols can be followed.Therefore, once such a process has been developed succesfully for acertain acylase it is very attractive to broaden the application of theacylase of choice instead of continuing the screening for enzymes fromother sources. Therefore enzyme modification through mutagenesis of theencoding wild type gene is thought to be superior to screeningespecially when small adaptation of the properties of the enzyme arerequired. Desired properties may include altered specificity, alteredspecific activity for a certain substrate, altered pH dependence oraltered stability. Mutagenesis can be achieved either by randommutagenesis or by site-directed mutagenesis.

Random mutagenesis, by treating whole microorganisms with chemicalmutagens or with mutagenizing radiation, may of course result inmodified enzymes, but then strong selection protocols are necessary tosearch for mutants having the desired properties. Higher probability ofisolating desired mutant enzymes by random mutagenesis can be achievedby cloning the encoding enzyme, mutagenizing it in vitro or in vivo andexpressing the encoded enzyme by recloning of the mutated gene in asuitable host cell. Also in this case suitable biological selectionprotocols must be available in order to select the desired mutantenzymes.

Site-directed mutagenesis (SDM) is the most specific way of obtainingmodified enzymes, enabling specific substitution of one or more aminoacids by any other desired amino acid.

The conversion of β-lactam intermediates to the desired semi-syntheticantibiotics may be performed chemically and enzymatically. If a suitableenzyme is available the enzymatic route is preferred because:

reactions can be performed stereospecifically;

reactants do not require side chain protection such as silylation;

less need for organic solvents, i.e. an organic solvent such asmethylene chloride can be omitted which reduces environmental problems;

compared to the chemical route usually less steps are required;

neither extreme temperatures nor pressures required;

usually lower content of byproducts.

Synthetic manipulation to produce the various penicillins andcephalosporins of choice basically starts from 6-APA, 7-ACA and 7-ADCA,respectively.

The enzymatic conversion takes advantage of the fact that any enzymaticreaction is reversible, if the correct conditions are applied. Theimportance of such applications has been highlighted in previousreviews. The literature gives several examples of the application ofpenicillin acylases in biosynthetic routes (J. G. Shewale et Al.,Process Biochemistry International, June 1990, 97-103). Acyl derivativesof 6-APA, 7-ADCA, 7-ACA, 3-amino-4-α-methyl monobactamic acid andpeptides have been prepared with side-chain moieties of varyingstructure. Besides 6-APA and 7-ADCA, penicillin acylase is used in theformation of antibiotic intermediates such as6-amino-2,2-dimethyl-3-(tetrazol-5-yl) penam, methyl-6-aminopenicillate,3-methyl-7-amino-3-cephem-4-carboxylic acid and 3-amino nocardic acid.The hydrolytic raction is catalysed at alkaline pH (7.5-8.5) while atacidic or neutral pH (4.0-7.0) it promotes acylation reactions.

Various factors affect the performance of an acylase in bioconversionprocesses:

reaction medium: pH, ionic strength, temperature, organic solvents,etc.;

enzyme stability with respect to process conditions;

reactant stability;

catalytic activity of the enzyme.

Except reactant stability which is not an enzyme property, the otherfactors may be a target for enzyme modification via protein engineering.

Various of these factors have been explored in order to makebiosynthesis processes economically viable. Methylesters which aresuperior acyl donors as compared to free acids of side chain acids havebeen used in the reaction. The equilibrium of the reaction has beenshifted in favour of acylation by changing the water activity around theenzyme molecule with certain solvents. E.g. polyethyleneglycol,methanol, ethanol, propanol, butanol, and acetone are used in enhancingthe yield of penicillin G, penicillin V and ampicillin.

Acylation reactions especially with 6-APA, 7-ADCA and 7-ACA generateantibiotics which are clinically important. However, the reaction needsto be monitored under strict kinetically controlled parameters. Althoughin some articles it was speculated that protein-engineering tools mightbe explored to obtain tailored enzyme molecules giving semisyntheticpenicillins and cephalopsorins at a yield competing with existingchemical processes, there was no teaching whatsoever neither how thisshould be carried out, nor which enzymes should be engineered, or whichamino acid residues should be substituted, nor any relation between thekind of substitution and the desired substrate.

The synthetic potential of a given penicillin acylase is limited due tothe specificity of the enzyme. Therefore, there is a substantialinterest in developing enzymes which are highly efficient indeacylation/acylation reactions to producedesired chemical entities. Ofparticular interest are the enzymatic deacylation of β-lactams(especially PenG, PenV, CefC, and derivatives thereof) to 6-APA and7-ACA and derivatives, and the acylation of the latter compounds toproduce semi-synthetic pencillins and cephalosporins of interest. Inaddition increased activity on more polar side chains or charged sidechains such as succinyl, glutaryl or adipyl is desired. In particular,it is of major importance to dispose of an efficient enzyme which iscapable of catalyzing the conversion of CefC (and derivatives) to 7-ACA(and derivatives).

Theoretical Aspects of the Application of Enzymes in Synthesis

Penicillin G acylases are hydrolases which catalyse the deacylation ofvarious β-lactam compounds. Moreover as enzymes catalyse reactions inboth directions, these acylases may also be used as a transferase tocatalyse the synthesis of condensation products such as β-lactamantibiotics, peptides, oligosaccharides or glycerides. Enzyme catalysedsynthesis may be carried out either as an equilibrium controlled or as akinetically controlled reaction.

In an equilibrium controlled process the enzyme only accelerates therate at which the thermodynamic equilibrium is established. The kineticproperties of the enzyme do not influence the equilibriumconcentrations. However, the thermodynamic equilibrium is dependent onreaction conditions such as pH, temperature, ionic strength, or solventcomposition. Often the conditions which favour the shift of thethermodynamic equilibrium in such a way that an optimal yield of thedesired product is obtained are usually not optimal for the performanceof the enzyme. In such cases enzyme engineering may be desired to adaptthe enzymes to conditions which are closer to the thermodynamic optimumof the reaction. In this aspect properties such as stability,temperature optimum and pH optimum may be useful targets.

In kinetically controlled reactions conditions are chosen in such waythat a considerable accumulation occurs of the desired product duringthe reaction under non-equilibrium conditions. In this case besides thealready mentioned parameters also the kinetic properties of the enzymeare an important factor in obtaining yields which can compete favourablywith existing chemical processes.

The kinetics of Penicillin G acylase. are consistent with catalysisproceeding via an acyl-enzyme intermediate. This intermediate plays akey role in the enzyme mechanism as is depicted in FIG. 1. In thisscheme the acylase acts as a hydrolase where the acyl group istransferred to water, or as a transterase where the acyl transfer froman activated substrate to a nucleophile is catalyzed. The chemicalentities are represented by general formulas. The nature of the chemicalentities X and Y in compound X--CO--NH--Y which are accepted as asubstrate by a particular acylase is determined by the specificity ofthat acylase. X represents the side chain, while Y represents the acylacceptor group. For instance, for the deacylation of PenG, X--CO--represents the phenyl-acetyl side chain and --NH--Y represents6-aminopenicillic acid. Given a certain enzymatic mechanism thespecificity is determined by the is architecture and the amino acidcomposition of the binding sites for X and Y.

In the first step of the mechanism, the substrate binds to the enzyme toform the non-covalent Michaelis-Menten complex. In the subsequent step,the covalent intermediate is formed between the enzyme and the acylmoiety of the substrate (E--CO--X). Formation of the acyl-enzyme mayoccur through cleavage of an amide bond (amide hydrolysis ofX--CO--NH--Y) or an ester bond (ester hydrolysis X--CO--O--R) and at lowpH it may also be formed directly from X--COOH. The nucleophile YNHbinds to the acyl-enzyme before deacylation. Under conditions whichfavour the deacylation (the enzyme acts as a deacylase or amidase) awater molecule will hydrolyse the acyl enzyme thereby liberating thesecond product X--COOH and regenerating the enzyme for a new catalyticcycle. Under conditions which favour formation of compound X--CO--NH--Y,the nucleophile Y--NH reacts with the acyl enzyme instead of water(aminolysis). For PenG the mechanism above was confirmed by theobservations that phenylacetic acid acts as a competitive inhibitor and6-APA as a non-competitive one.

In general the formation of the acyl-enzyme from amides (v₁) is slowcompared to the hydrolysis of the acyl enzym (v₃). However, when theappropriate ester derivatives of the side chain are used (X--CO--O--R)or just the amide (X--CO--NH2) then the formation of the acyl-enzyme(v₂) is relatively fast in comparison with hydrolysis (v₃). Theconsequence is that the acyl enzyme intermediate will accumulate. In thepresence of suitable compounds with a free primary amino group (generalrepresentation Y--NH2) such as, for example, 6-APA, 7-ACA, 7-ADCA whichare bound by the acylase, an amide bond may be formed giving X--CO--N--Y(v₋₁, aminolysis).

With respect to the preference for chemical entities X and Ysubstitution of residues in the binding sites for X and Y at the enzymealter this preference. Changes in substrate specificity include allcombinations of increase and decrease of V_(sax) and K_(s). In somecases a more specific enzyme is required, e.g. with mixtures ofenantiomers it may be useful when the enzyme is selective for only oneof the enantiomers. In other cases, e.g. the conversion of rather purecompounds, a higher conversion rate might be preferred at the cost ofselectivety. At high substrate concentrations a higher V_(sax) ispreferred while Km is less important.

Acylases used for substrate activation and kinetically controlledsynthesis may be altered in such a way that their catalytic ability tohydrolyse compounds (V₃ =transfer acyl group to water) has beensuppressed with respect to acyl transfer to a non-aguous acceptornucleophile (v₋₁): ratio V₋₁ /V₃ increased relative to wild type.

The ratio of transferase to hydrolase activity is the enzyme propertythat influences yield in kinetically controlled synthesis ofcondensation products. The ratio of the apparent second order rateconstants for the acyl transfer to YNH or H20 can be determined from theinitial rates of formation of X--CO--NH--Y and X--COOH from theacyl-enzyme.

Transferase activity may be improved by improving the affinity of thenucleophile for the enzyme-acyl complex with respect to water. As thetransfer of the acyl group (v₋₁) is proportional to amount ofnucleophile bound to the acyl-enzyme an increased affinity for theenzyme-acyl complex will improve the yield of the condensation productwith respect to hydrolysis.

In addition a higher yield in an enzyme catalysed biosynthesis may beobtained by reducing the hydrolysis of the desired products (v₁ v₃).Variants for which the hydrolysis of amide bonds relative to ester bondshas been decreased are still able to form the acyl enzym from estersubstrates (v₂) but have relatively weak hydrolysis activity for theproduct amide bond (increased ratio v₁ /V₂ with respect to wild type).

Relevant Literature

Several genes encoding Type-IIA Penicillin G acylases have beensequenced, viz. the genes from E. coli (G. Schumacher et al., NucleicAcids Research 14 (1986) 5713-5727), Kluyvera citrophila (J. L. Barberoet al., Gene 49 (1986) 69-80), Alcaligenes faecalis (U.S. Pat. No.5,168,048, Gist-brocades), Providencia rettgeri (G. Ljubijankic et al.,J. DNA Sequencing and Mapping 3 (1992) 195-201) and Arthrobacterviscosis (M. Konstantinovic et al., (1993) EMBL databank entry L04471).

The use of recombinant DNA methods has enabled an increase of theproduction levels of commercially used penicillin acylases (Mayer etal., Adv. Biotechnol. 1 (1982) 83-86) and has enlarged the insight intothe processing of these enzymes (G. Schumacher et al., Nucleic AcidsResearch 14 (1986) 5713-5727). The penicillin acylase of E. coli wasfound to be produced as a large precursor protein, which was furtherprocessed into the periplasmic mature protein constituting a small (α)and a large (β) subunit. Cloning and sequencing of the Kluyveracitrophila acylase gene has revealed a close homology with the E. coliacylase gene (J. L. Barbero et al., Gene 49 (1986) 69-80). Also forProteus rettaeri (G. O. Daumy et al., J. Bacteriol. 163 (1985)1279-1281) and Alcaligenes faecalis (U.S. Pat. No. 5,168,048 andEP-A-453048, Gist-brocades) Penicillin G acylase a small and a largesubunit has been described.

These publications neither teach nor suggest the instant invention.

Redesigning of specific activity of enzymes with the aid ofprotein-engineering techniques has been described.

Patent applications EP-A-130756 and EP-A-251446 describe the selectionof residues and the mutagenesis of some of these residues in a certaingroup of serine protease with the purpose to alter the kineticproperties of these enzymes.

As these patent applications specifically deal with a certain type ofserine proteases (the subtilisin type), these publications do notindicate which residues modulate the catalytic properties of Type-IIaPenicillin G acylases.

Wells et al. (Proc. Natl. Acad. Sci. USA 84 (1987) 5167) show an examplefor subtilisin. Bacillus licheniformis and B. amyloliguefaciens serineprotease differ by 31% (86 residues) in protein sequence and by a factorof 60 in catalytic efficiency on certain substrates. By substituting 3of the 86 different amino acids from the B. amyloliguefaciens sequenceby the corresponding B. licheniformis residues the catalytic activity ofthe mutant enzyme was improved nearly 60 fold.

Wilks et al. (Science 242 (1988) 1541) describe how a lactatedehydrogenase was changed into a malate dehydrogenase by mutatingglutamine 102 into arginine 102. In both cases, serine protease andlactate dehydrogenase, the inspiration for the modification proposalcame from comparison with naturally occuring enzymes, which alreadyshowed the desired specificity. In the same way the specificity ofcytochrome p450₁₅α was changed into the specificity of cytochromep450_(coh) by replacing Leu209 with Phe209 (Lindberg and Negishi, Nature339 (1989) 632).

Patent application WO93/15208 describes a method for modifying thespecificity and or efficiency of a dehydrogenase while retaining itscatalytic activity, characterized in that it comprises: selecting anenzyme, the tertiary structure of which is substantially known ordeduced; identifying at least one specificity and/or efficiency-relatedregion; identifying or constructing unique restriction sites boundingthe identified region in the DNA encoding therefor; generating a DNAsequence which corresponds to at least a portion of the identifiedregion, except that the nucleotides of at least one codon arerandomized, or selecting as a substitute for at least a portion of theidentified region an alternative such region, which may itself besimilarly randomized; using the generated or substitute DNA sequence toreplace the original sequence; axpressing the DNA including thegenerated or substitute DNA sequence; and selecting for a desiredmodification so that the DNA coding therefor may be isolated. Asdehydrogenases are in no way related to Penicillin G acylase, thispatent application does not reveal the residues in the acylase whichshould be substituted to alter its kinetic properties.

Forney et al. (Appl. and Environm. Microbiology 55 (1989) 2550-2556;Appl. and Environm. Microbiology 55 (1989) 2556-2560) have isolated bycloning and in vitro chemical/UV random mutagenesis techniques E. colistrains capable of growing on glutaryl-L-leucine orD(-)-α-amino-phenyl-acetyl-(L)-leucine. Penicillin acylase produced bythe mutants hydrolyse glutaryl-L-leucine between pH and 6 orD(-)-α-amino-phenyl-acetyl-(L)-leucine at pH 6.5. Although it issupposed that the specificity shift of the Penicillin G acylase is dueto one or more mutations in the acylase, the residue(s) involved nor thekind of mutation(s) were identified.

J. A. Williams & T. J. Zuzel (Journ. of Cell. Biochem. (1985) supplement9B, 99) reported in an abstract of a poster presentation themodification of the substrate specificity of Penicillin G acylase by invitro mutagenesis of a methionine. Although the abstract does not reportthe position of this methionine, from the poster it seemed to bepossible to conclude that it involved position Met168 in E. coliacylase. However, this work did not reveal any details how substitutionof this methionine relates to 25 the observed specificity change. Prietoet al. (I. Prieto et al., Appl. Microbiol. Biotechnol. 33 (1990)553-559) replaced Met168 in K. citrophila for Ala, Val, Asp, Asn, Tyrwhich affected the kinetic parameters for PenG and PenV deacylation. Inaddition mutants Lys375Asn and His481Tyr were made which showed hardlyany effect on k_(cat) /Km.

J. Martin et al. analysed mutant Met168Ala in K. citrophila penicillinacylase and reported altered kinetic properties. (J. Martin & I. Prieto,Biochimica et Biophysica Acta 1037 (1990), 133-139). These referencesindicate the importance of the residue at position 168 in E. coli and K.citrophila for the specificity with respect to the acyl moiety. However,this work did not reveal any details how substitution of this methioninerelates to the specificity change for the conversion of a desiredsubstrate.

Wang Min reported mutagenesis of Ser177 in E. coli Penicillin G acylaseto Gly, Thr, Leu, Arg but failed to obtain active acylases. (Wang Min etal., Shiyan Shengwu Xuebao hi (1991), 1, 51-54).

Kyeong Sook Choi et al. (J. of Bacteriology 174 (1992) 19, 6270-6276)replaced the β-subunit N-terminal serine in E. coli penicillin acylaseby threonine, arginine, glycine and cysteine. Only when the N-terminalresidue was cysteine the enzyme was processed properly and a matureenzyme but inactive enzyme was obtained. In addition chemicalmutagenesis of the β-subunit N-terminal serine also led to severe/almostcomplete loss of activity (Slade et al., Eur. J. Biochem. 197 (1991)75-80; J. Martin et al., Biochem. J. 280 (1991) 659-662).

Sizman et al. (Eur. J. Biochem. 192 (1990) 143-151) substituted serine838 in E. coli for cysteine without any effect on the post-translationalprocessing nor on the catalytic activity of the enzyme. In additionSizman et al. made various deletion mutants of penicillin acylase. Itshowed that correct maturation of the acylase is very sensitive tomutagenesis. All β-subunit C-terminal deletion mutants were notexpressed except for the mutant lacking the last three residues which,however, was very unstable. Insertion of four residues in E. coli atposition 827 also failed to give active enzyme.

Prieto et al. replaced glycine 310 in Kluyvera citrophila penicillinacylase for glutamic acid. However, no active enzyme was obtained.

In EP-A-453048 it has been described how protein engineering may be usedto alter the specificity of Type-IIa as well as Type-IIb acylase.However, the applied procedures are limited to the generation oflibraries of randomly generated acylase mutants which have to bescreened for a desired activity. Although by the method described inthat patent application the number of amino acid positions which may bemutated has been reduced, the number of remaining positions is stilllarge, so that position directed mutagenesis would be a laborious job.The present invention, however, gives a much more limited number ofpositions which are to be mutated. In addition amino acids at thesepositions are in direct contact with the substrate, which means thatsubstitution will affect interaction with the substrate directly.Moreover the procedure leading to the present invention allows one tochoose a particular amino acid substitution in order to obtain a desiredeffect for a specific substrate.

SUMMARY OF THE INVENTION

The present invention provides an isolated mutant prokaryotic PenicillinG acylase or its preenzyme or preproenzyme comprising:

a substitution at one or more selected sites of the positionscorresponding to A139 to A152, B20 to B27, B31, B32, B49 to B52, B56,B57, B65 to B72, B154 to B157, B173 to B179, B239 to B241, B250 to B263,B379 to B387, B390, B455, B474 to B480 in Alcaligenes faecalisPenicillin G acylase or its pre- or preproenzyme; and

an altered substrate specificity or altered specific activity relativeto the corresponding wild-type unsubstituted Penicillin G acylase.Preferably, said isolated mutant prokaryotic Penicillin G acylase isoriginated from Alcaligenes faecalis.

Furthermore a nucleic acid sequence encoding said mutant acylase, avector which comprises said nucleic acid sequence, and a microorganismhost strain transformed with said vector have been provided for by thepresent invention.

According to another aspect of the invention a process of preparing saidisolated mutant Penicillin G acylase has been provided, which processcomprises:

culturing a microorganism host strain transformed with an expressionvector comprising a nucleic acid sequence encoding a mutant acylaseenzyme as defined above, whereby said mutant acylase is produced; and

isolating said acylase.

Finally, a method for conducting an acylation or deacylation reactionhas been provided, said process comprising contacting said isolatedmutant Penicillin G acylase with a substrate for said acylase underconditions suitable for said reaction to occur. Preferably, a β-lactamcompound is produced by said process.

Especially, a method for deacylating an acylated 6-amino penicillanicacid, an acylated 7-amino(desacetoxy)cefalosporanic acid or a salt orester thereof to form the corresponding 6-amino penicillanic acid or7-amino(desacetoxy)cefalosporanic acid or salt or ester thereof,respectively, which comprises contacting said 6-acylated or 7-acylatedcompound with a mutant acylase as defined above under conditionssuitable for deacylation to occur, and a method for producing asemi-synthetic acylated 6-amino penicillanic acid, an acylated7-amino(desacetoxy)cefalosporanic acid or a salt or ester thereof whichcomprises contacting a corresponding 6-amino or 7-amino β-lactam and anacylating agens with a mutant acylase as defined above under conditionssuitable for acylation to occur, has been provided for.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Reaction scheme for Type-IIa penicillin acylases catalysedconversions. EH represents the enzyme where H stands for the protonwhich is transferred to the leaving group. X stands for the acyl moietyor side chain. Y is the compound to be acylated (acylation) or to bedeacylated (deacylation). Compound X--CO--OR may also be a simple amideX--CO--NH2.

FIG. 2: Alignment of α(2a) (FIGS. 2A-2B) and (26)(FIGS. 2C-2F) subunitof Type-IIa penicillin acylases mature enzymes. Alcaligenes faecalis(afae), SEQ ID NO:27 and SEQ ID NO:32, respectively), E. coli (ecol; SEQID NO:28 and SEQ ID NO:33 respectively), Kluyvera citrophila (kcit; SEQID NO: 29 and SEQ ID NO: 34, respectively), Arthrobacter viscosus (avis;SEQ ID NO: 30 and SEQ ID NO: 35, respectively). Providencia rettgeri(pret) Providencia rettgeri (pret; SEQ ID NO: 31 and SEQ ID NO: 36,respectively). Chain identifier A and B for α and β chain, respectively.An asterix denotes that the sequence contains the same amino acid atthat position as the sequence from the A. faecalis acylase. For theProvidencia rettgeri acylase the N-terminus and the C-terminus of thea-subunit not known. N-terminus β subunit Providencia rettaeri based onalignment with other acylases.

FIG. 3: Atom names PenG referring to nomenclature used in tables 2 and3.

FIG. 4a: Stereo picture of the active site A. faecalis PenG acylasearound phenylacetyl moiety.

FIG. 4b: Stereo picture of the active site A. faecalis PenG acylasearound the 6-ACA moiety.

FIG. 5: pMcAF mutagenesis vector with A. faecalis Penicillin acylasegene, E. coli ori `high` copy, Tac promotor, Fd-termintor, cap⁻, amp⁸f1-origin.

FIG. 6: Maximal deacylation velocity of wild type A. faecalis acylaseand the mutants B:L56G, B:L56A, B:L56V, B:I177V, B:I177S, B:A67S, B:A67Gfor various substrates. Velocities for each variant are relative toPenG: V_(sax) (X)/V_(sax) (PenG). X represents PenV, CefG, Ampicillin(Ampi), (D)Phenylglycinamide ((D)PGA) or NIPAB.

FIG. 7: The maximal deacylation velocity of wild type A. faecalisacylase and the mutants A:M143V, B:L56K, A:F147Y for various substrates.Velocities for each variant are relative to PenG: V_(sax) (X)/V_(sax)(PenG). X represents PenV, CefG, Ampicillin (Ampi), (D)Phenylglycinamide((D)PGA) or NIPAB.

DETAILED DESCRIPTION OF THE INVENTION

Hydrolysis/deacylation.

The present invention relates to the identification of residues whichalter the kinetic properties of Penicillin G acylase, whereby saidresulting Penicillin G acylase variant is more useful than saidprecursor Penicillin G acylase for the deacylation of primaryaminogroups such as, for example, occur in penicillins andcephalosporins. These kinetic properties comprise specific activity, pHdependence of kinetic parameters, substrate specificity,stereo-selectivity and the ratio transferase to hydrolase activity.

Synthesis/acylation.

The present invention relates to Penicillin C acylase variants derivedfrom precursor Penicillin G acylases via recombinant DNA methodology bychanging at least one amino acid residue in said precursor, saidPenicillin G acylase variant being more useful than said precursorPenicillin G acylase for the acylation of primary amino groups such as,for example, occur in β-lactam nuclei (preparation of semi-syntheticβ-lactam compounds) and peptides.

The present invention relates to Penicillin G acylase variants derivedfrom precursor Penicillin G acylases via recombinant DNA methodology bychanging at least one amino acid residues in said precursor, saidPenicillin G acylase variant being characterized by having a higherratio transferase to hydrolase activity than said precursor Penicillin Gacylase.

The Penicillin G acylases which are subject of this invention:

are isolated from prokaryotes;

are transcribed as a single peptide chain precursor;

are processed intracellularly after transcription resulting in aheterodimer with a small N-terminal domain (the α-domain) and a largerC-terminal domain (the β-chain). The molecular weight of the N-terminaldomain is in the range 16-28 kDa. The molecular weight of the C-terminaldomain is in the range 54-68 kDa;

may occur in solution as multimers of the αβ hetero-dimers;

have a serine at the N-terminus of the β-subunit.

Examples of such acylase producing microorganisms are certain strains ofthe species Escherichia coli, Kluyvera citropthila, Providenciarettgeri, Pseudomonas sp., Alcaligenes faecalis, Bacillus mecaterium,and Arthrobacter viscosus.

Several genes encoding Penicillin G acylases have been sequenced, viz.the genes from E. coli, Kluyvera citrophila Alcaligenes faecalis,Proteus rettgeri and Arthrobacter viscosis.

The alteration of the substrate specificity of Penicillin G acylases isachieved in such a way that the mutant enzymes are able to cleave orsynthesize penicillin and cephalosporin derivatives possessingside-chains other than phenylacetyl, which is the natural side-chain ofpenicillin G. Examples of side-chains which are presently notsignificantly affected by Penicillin G acylases are acyl groups derivedfrom the dicarboxylic acids succinic acid, glutaric acid, adipic acidand aminoadipic acid (the latter being the natural side-chain of CefC).

In another aspect the alteration of the specificity and activity ofPenicillin G acylases is performed for side-chains which are alreadyexisting substrates for the said acylases. Using protein engineering theaffinity for a substrate can be altered (e.g. increased, expressed by alower K_(s) for said substrate), the catalytic turnover may be altered(e.g. increased, expressed by a higher k_(cat) for said substrate) orthe second order rate constant may be altered (e.g. expressed by analtered k_(cat) /Km ratio, a parameter (which is usually used to comparespecificity of an enzyme for different substrates). Relevant substratesin this aspect include acylated β-lactam derivatives such as penicillinV (PenV), ampicillin, amoxicillin, cefalexin, cefadroxyl or cefaclor.Moreover alteration of kinetic properties with respect to simple amidesand esters of the acyl moiety are useful for obtaining increasedaccumulation of the acyl enzyme intermediate which may improve the yieldin biosynthesi-s processes.

In another aspect the alteration of the specificity and activity ofPenicillin G acylases is performed in order to increase the stereospecificity of Penicillin G acylases which results in enzymes which showimproved enantiomeric excess in conversions with racemic mixtures ofchiral compounds. Such property makes the Penicillin G acylase extremelyuseful for synthesis of enantiomerically pure semisynthetic antibioticsfrom racemic mixtures of phenylacetyl side chains or activatedderivatives of the phenylacetyl side chains (e.g. phenylglycine-amidesor esters therefrom, p-hydroxyphenylglycine-amides or esters therefrom,etc.) containing a chiral α-carbon due to the presence of an amino group(e.g. ampicillin, cefalexin, amoxicillin, cefadroxyl, cefaclor) or ahydroxyl group (cefamandol).

Apart from stereoselectivity for the acyl Cα position Penicillin Gacylase exhibits also stereoselectivity for the amino part of thesubstrate. In case of amino acids the acylase requires theL-configuration at the Cα atom. In another aspect of the inventionsteroselectivity of the enzyme for the amino part of the substrate maybe altered.

In another aspect of the invention the product inhibition is reducedwith respect to the wild type enzyme. The desired variant maintains itsinitial high deacylation rate for a longer period during conversionresulting in a higher productivity. Examples of such inhibitory productsare phenylacetate, phenoxyacetate, phenylglycine, p-hydroxyphenylglycineetc.

In another aspect of the invention the transferase activity of theenzyme is improved with respect to the hydrolases activity which makesthe enzyme more useful in biosynthetic conversions. In particularvariants with improved performance in the enzymatic synthesis ofamoxicillin, ampicillin, cefaclor, cefadroxil, cefprozil, cephalexin,and cephradine are preferred embodiments.

Compared to the precursor acylase desired variants for biosynthesis aremore easily deacylated by a β-lactam nucleus than by water (ratioaminolysis/hydrolysis). This may be obtained by improving the binding ofthe nucleophile relative to water Desired variants have alteredesterase/amidase ratio for particular substrates relative to theprecursor enzyme i.e. for certain side chains the desired enzyme-shows adecreased amidase activity for amide derivatives of those side chainscompared to the esterase activity for esters of the corresponding sidechains.

In order to achieve alterations in the enzyme molecule, it is highlydesirable to avail of the 3D structure of said enzyme. Sofar, nohigh-resolution, 3D-structures of acylases have been published.

The known Penicillin G acylase gene sequence derived amino acidsequences were aligned in such a way that optimal homology was obtained.For sequence alignment the types of amino acids may be suitably used asparameters, based on identity but also on similarity. For example,serine is similar to threonine, aspartic acid is similar to glutamicacid, etc. The results are shown in FIG. 2 which gives an alignment ofPenicillin G acylases from Escherichia coli, Kluyvera citrophila,Alcaligenes faecalis, Providencia rettgeri and Arthrobacter viscosis.The alignment of the five amino acid sequences reveals a significanthomology between the Penicillin G acylases which points to a similar3D-structure.

In an embodiment of the invention corresponding positions of otherPenicillin G acylases, which are structurally homologous to Alcaligenesfaecalis Penicillin G acylase can be substituted in the same way asAlcaligenes faecalis at the positions which are homologous to thepositions in Alcaligenes faecalis Penicillin G acylase. Thecorresponding positions for these proteases may be obtained from theamino acid alignment as depicted in FIG. 2. In FIG. 2 the amino acidsequence of various acylases have been aligned, with respect to thesequence of the acylase of Alcaligenes faecalis (A. fae).

Although the selection of residues will be demonstrated here using thespecific example of Alcaligenes faecalis Penicillin G acylase it isclear that due to homology similar substitution sites can be selected inPenicillin G acylases obtained from other species. The approachdescribed would give rise, after amino acid replacement at correspondingpositions in the Penicillin G acylase from the other species, to similaraltered kinetic properties of other Penicillin G acylase also. Bysimilar is meant the kind of effect which the substitutions have on thekinetic parameters change.

In an embodiment of the invention genes encoding known Penicillin Gacylases, for example, Penicillin G acylases from Escherichia coli,Kluyvera citrophila, Alcaligenes faecalis, Providencia rettgeri andArthrobacter viscosis or any other organism producing such enzymes, aremutated in such a way that the enzymes obtain an altered specificity fortheir substrates.

In an embodiment of the invention, genes encoding the structurallyhomologous Penicillin G acylases, for example, Penicillin G acylasesfrom Escherichia coli, Kluyvera citrophila, Alcaligenes faecalis,Providencia rettgeri and Arthrobacter viscosis, are mutated in such away that the enzymes obtain an altered substrate specificity or newspecificity.

Changes in substrate specificity demonstrated in the present inventioninclude all combinations of increase and decrease of V_(sax) and K_(s)for both penicillin and cephalosporin derivatives. A person skilled inthe art will understand that this encompasses the changes in otherkinetic parameters. Furthermore, the specificities for other substratewill inherently be changed also. The proposed rules for changing thesubstrate specificity are not restricted to the mentioned substrates,they can be applied to other substrates among these are phenylacetyl orphenoxyacetyl derivatives of amino acids, aminoalkylphosphonic acids,primary and secondary alcohols, cefamicines, nocardicines, monobactams,nucleic acids, carbohydrates, peptides.

As the mechanism of maturation of Penicillin G acylase from aone-peptide chain to an active dimer is still obscure another importantaspect of the invention shows that it is possible to replace active siteresidues in Penicillin G acylase without affecting the maturation of theacylase.

The underlying invention to provides a methods to recruit novelspecificities for Type-IIa Penicillin G acylases. For the introductionof point mutations a rational approach is taken, relying on theapplication of protein crystallography, molecular modelling andcomputational methods, enzymology and kinetics, molecular biology andprotein chemistry techniques. The strategies for the identification oftargeted mutations in Penicillin G acylase are innovative in a sensethat it is recognized that point mutations may affect several differentproperties of the protein structure at once. In particular some pointmutations may prevent proper folding or correct processing resulting inan inactive enzyme. Therefore, although the described strategies makeuse of well established structure-function relationships, they alsoprovide a rational way to avoid or correct unwanted alterations ofsecondary properties.

According to the present invention specific amino acid positions to besubstituted have been identified within the available 753 positions inthe Penicillin G acylase molecule from A. faecalis, and the effect ofsuch mutations on the particular properties of the enzyme. Thus A139 toA152[SEQ ID NO:27], B1, B2, B20 to B27, B31, B32, B49 to B52, B56, B57,B65 to B72, B154 to B157, B173 to B179, B239 to 241, B250 to B263, B379to B387, B390, B455, and [SEQ ID NO:32] B474 to B480 are identified asimportant positions with regard to the catalytic properties of theenzyme. various specific residues have been identified as beingimportant with regard to substrate specificity. These residues include:A:Met143, A:Arg146, A:Phe147, A:Thr150 [SEQ ID NO:27], B:Pro22, B:Phe24,B:Gly25, B:Tyr27, B:Tyr31, B:Thr32, B:Pro49, B:Tyr52, B:Leu56, 3: Phe57,B:Gly66, B:Ala67, B:Thr68, B:Ala69, B:Gly70, B:Pro71, B:Trp154,B:Val157, B:Met173, B:Ile175, B:Ser176, B:Ile177, B:Trp179, B:Asn239,B:Trp240, B:Thr251, B:Thr253, B:Tyr254, B:Tyr255, B:Trp256, B:Arg261,B:Met262, B:Asn379, B:Pro380, B:Gly381, B:Ser382, B:Ile383, B:Asn384,B:Lys390, B:Phe455 B:Thr477, B:Glu478 [SEQ ID NO:32]. The identificationof these positions, including those yet to be mutated is based on a 3Dmodel of the A. faecalis Penicillin G acylase (see FIGS. 4a and 4b).

Selection procedure for residues which alter desired properties.

Desired rrorerties are altered catalvtic properties, alteredspecificity, improved transferase activity

The crucial first step for performing site-directed mutagenesis with theobject to alter kinetic properties of an enzyme is to obtain a 3Dstructural model of the subject Penicillin G acylase complexed with theβ-lactam compound of interest. This can be done in two ways, namely viaa direct experimental approach, or via an indirect approach usingmolecular modeling.

The direct approach.

Determine the 3D-structure of the subject Penicillin G acylase incomplex with the β-lactam compound of interest by X-ray diffraction.However, when the particular β-lactam compound is a substrate for theparticular Penicillin G acylase, it will be converted into the productsof the reaction in the time-course of the structure determinationexperiment. In such cases cryo-crystallography may be applied or veryfast data-collection techniques such as Laue diffraction. Withconventinal techniques binding of fragments of the substrate can revealthe binding site. As an alternative the substrate can be modified insuch a way that the scissile bond in the substrate cannot be cleaved bythe enzyme (e.g. phosphoamide or phosphonate bonds instead of a peptidebond in a peptide, D. E. Tronrud et al. Science 235 (1987) 571-574).However, an elegant method is to replace one or more of the catalyticresidues resulting in an inactive enzyme which cannot convert thesubstrate but can still bind the substrate. For example, in Penicillin Gacylase the β-subunit N-terminal serine may be mutated to cysteine. Whenit is not possible to obtain a 3D structure of the subject acylasecomplexed with the desired β-lactam derivate by experiment, conventionalcomputer modelling techniques can be applied. Chemical modificationstudies and site directed mutagenesis revealed the N-terminal serine ofthe β-subunit to be critical for catalytic activity. Surprisinglycalculation of the accessible residues in A. faecalis Penicillin Gacylase model revealed a deep hydrofobic cavity near the β-subunitN-terminal serine which accomodates the Penicillin G phenylacetyl sidechain perfectly while positioning the β-subunit N-terminal serine in anideal position for nucleophilic attack at the peptide carbonyl of PenG.

In the next step the β-lactam moiety was positioned while keeping thephenylacetyl group fixed in its binding pocket. Atomic overlap betweensubstrate and enzyme is avoided as much as possible while positiveinteractions are maximized. Relevant positive interactions whichcontribute to binding are hydrogen bonding, electrostatic interactionsand favourable VanderWaals contacts. The contribution of hydrofobicinteractions can be estimated from the calculation of the accessiblenon-polar surface which is buried by binding the substrate to theenzyme.

In addition to manual manipulation of the substrate computationaltechniques are applied to optimize the substrate-enzyme complex.Molecular mechanics techniques such as energy minimization and moleculardynamics are very useful. Suitable forcefields for proteins such asCVFF, AMBER, CHROMOS may be used.

The final model is used to survey the environment of the PenG molecule.This survey supplies crucial insight in the residues which interact withthe PenG molecule (see example 1). In addition it provides insight whichresidues interact with which parts of the substrate. This informationprovides the molecular biologist with only a limited set of residuescompared to the overall size of the acylase (753 residues) which can beused to modulate the catalytic properties of Penicillin G acylase. Now aperson skilled in the art of site specific mutagenesis just has to focuson only a limited number of residues, substitute these residues andselect for desired altered catalytic properties.

In general when a substrate binds to the free enzyme it causes lo somestrain in the enzyme and in the substrate. Such strain can be relievedby molecular mechanics calculations allowing atoms to shift positionwith respect to each other. Comparison of the enzyme-substrate complexwith the free enzyme will indicate which residues are affected most bysubstrate binding. Parameters which are important in this aspect are RMSpositional shifts of residues with respect to the free enzyme, changesin the electrostatic environment around residues with respect to thefree enzyme, hydrogen bond formation or the change of free energy ofresidues. Electrostatic potentials may be calculated using a programsuch as DELPHI (Biosym Technologies). As residues which are affected bybinding of the substrate will in turn affect the binding of thesubstrate, substitution of these residues is a preferred embodiment ofthis invention taking into account the restrictions for substitution ofamino acids in proteins structures. Substitution that should be avoidedare those substitutions which are expected to affect typical structuralarrangements such as: salt bridges, packing of helices, stabilization ofhelices by keeping a negative charge at the start of a helix, initiationof helices, e.g. prolines at the start of a helix, Phi-psi angles whichare outside the allowed region for the residue that is going to beinserted.

The proposed rules for changing the activity for a certain substrate arenot restricted to PenG, they can be applied to other substrates as well.For example, instead of PenG a cephalosporin molecule may be taken suchas CefG, which has the phenylacetyl side chain in common with PenG. Inthis case the whole modelling procedure may be repeated as describedabove. However, we prefer to substitute in the computer the 6-APA moietyof the PenG molecule which is complexed to the Penicillin acylase forthe 7-ADCA moiety and subsequently refine the structure by molecularmechanics. Comparison of the structures of Penicillin G-acylase complexwith the CefG-acylase complex will establish the residues which havebeen affected by modification of the substrate. Residues which areaffected by modification of PenG will in turn modulate the binding ofthe modified substrate. Substitution of such residues is a preferredembodiment in order to alter the kinetic properties of such a modifiedsubstrate with respect to PenG.

For some modifications of the substrate it turns out to be impossible torelieve the strain caused by the modification without effecting theposition of the scissile peptide bond with respect to the β-subunitN-terminal serine nucleophile. In such cases the distance from theβ-subunit N-terminal serine nucleophile to the carboxyl carbon of thescissile bond is constrained within the range 2 to 3 Å during energyminimization and molecular dynamics. In addition computationalmutagenesis of the acylase is performed to reduce undesirableinteraction with the substrate and increase benificial interaction(relevant interactions have been discussed above). However, when thebinding of the modified substrate is unwanted and should be prohibited,undesirable interaction may even be increased at such positions by sitedirected mutagenesis. This approach establishes a limited number ofmutations which will alter the kinetic properties in a desireddirection. Subsequently such limited number of mutations can be made andtested for the desired properties.

Desired modifications imply substitution of the PenG side chain benzenering by a five- or six-membered hydrocarbon ring (e.g. cyclohexadienyl,cyclohexenyl, cyclohexyl), optionally substituted either by afive-membered heterocycle containing one to four heteroatoms (N, O, orS) (e.g. thienyl, furyl) which heterocycle may be optionallysubstituted, or by an aliphatic side chain (e.g. propyl, butyl, pentyl,heptyl) which may be optionally substituted. Side chains may have one ormore substituent including but not limited to hydroxyl, halogen, alkyl,alkoxyl, carboxyl, nitro, amino, and the like. In addition thephenylacetyl side chain may be substituted at the a-position resultingin a D- or L-stereoisomer. Substituent may include but are not limitedto hydroxyl, halogen, alkyl, alkoxyl, carboxyl, nitro, amino, and thelike. Selecting residues which affect the selectivity of the acylasewith respect to stereoisomers is a preferred embodiment of theinvention. Examples of desired side chains are, for example,2-thienylacetyl, α-hydroxyphenylacetyl, p-hydroxyphenylacetyl,p-hydroxyphenylglycyl, phenylglycyl, succinyl, glutaryl, adipyl,α-aminoadipyl etc.

Beside modification of the β-lactam side chain also the β-lactam moietyitself may be subject to modification. As exemplified above the 6-APAmoiety may be replaced by 7-ADCA. Instead 7-ACA may be taken. Inaddition the β-lactam moieties may be substituted at one or morepositions. In particular the cephalosporins may contain substituents atthe sulphur, at the 3-position or at the 4 position. For example, the3-position may be substituted with a halogen atom, a methoxy, a methylor a methylene bonded via a bridging atom O, N, or S to an organicmoiety or five- or six membered (hetero) cyclic group which mayoptionally be substituted. At the 4-position the carboxylic acidsubstituent may be modified with various carboxyl protecting groups.Furthermore the given method allows also to analyze the structuralrequirements for acylases which may convert β-lactam moieties such ascarbapenems, nocarcidines, monobactams or derivatives derived therefrom.

For the purpose of biosynthesis the interaction of the acylase withreactive derivatives of desired side chains may be modulated. Usefulexamples of such side chain derivatives are alkyl esters, amides andacylated amino acids.

The process of the invention can be used to select those position intype-II Penicillin G acylases at which amino acids should be substitutedin order to affect the interaction with penicillins/cephalosporins andtheir derivatives which results in enzymes with altered kineticproperties. Position directed mutagenesis will provide a limited numberof variants which can be easily tested for improved conversion of thedesired substrate. This in contradiction to the random approach whichresults in an enormous number of mutants which is very difficult tohandle.

Materials and methods

Mutagenesis.

For the construction of mutant acylase genes the overlap extensionpolymerase chain reaction has been used essentially as described by Hoet al. (Gene 77 (1989) 51-59). Mutant oligo's were used in combinationwith flanking oligo's to generate DNA amplification products harbouringthe desired mutation. This mutant DNA fragment was exchanged with acorresponding restriction fragment of the wild type gene, e.g. pMcAF.The mutant oligo's have been designed to harbour single and multiplemutations.

Site-directed mutagenesis of cloned DNA fragments can also be carriedout as described by Stanssens (Stanssen et al., Nucleic Acids Res. 17(1989) 4441-4454) with the aid of the phasmid pMa/c system. Suitablegapped duplex molecules of acylase genes were constructed. With specificmismatch oligonucleotides site directed mutations were introduced.Expression of acylase genes was obtained in E. coli WK6 either from thehomologous expression signals or from the E. coli lac, tac or trppromoter (De Boer et al., Proc. Natl. Acad. Sci. USA 80 (1983) 21-25).`Spiked` oligo mutagenesis and random mutagenesis of the gapped DNA wasperformed as described (EP-453048).

Both PCR overlap extension and gapped duplex have been combined withanother type of mutagenesis: targeted random mutagenesis (TRM).

This comprises the inclusion of two or more bases at the codon for aspecific amino acid during the synthesis of the oligonucleotide. Indoing so, a mutagenic oligonucleotide which can generate all otherpossible amino acids at a chosen codon can be synthesized. A singleamino acid position or a combination of several positions can bemutagenized in that way.

Selective media.

Selective media for phenylacetyl L-leucine (`fal` ) were prepared asdescribed by Garcia (Garcia et al., J. Biotech. 3 (1986) 187-195).Minimal plates are as follows: M63 minimal agar, 2 g/l glucose, 1 mg/lthiamine, 10 mg/i L-proline and the appropriate antibiotic (50 μg/mlchloramphenicol (cap) or 25 μg/ml ampicillin (amp)). For selections onside-chain specificity (e.g phenylacetyl, phenoxyacetyl, phenylglycyl,p-hydroxyphenylglycyl, adipyl or α-aminoadipyl) of acylases 100 μg/l ofthe corresponding acyl L-leucine was included into minimal plates.Transformants or mutants of E. coli HB101 (Leu) growing exclusively inthe presence of the acyl L-leucine are considered to harbour an acylasegene with the desired specificity. Instead of leucine the amino acidmoiety of the selective substrate may also be varied. In such case asuitable auxotrophic mutant of E. coli was used for selection. Forexample, selection on the substrate N-adipyl-L-leucine was carried outwith E. coli strain PC2051 as a host (obtained from Phabagen, Utrecht,the Netherlands). The special screenings substrates were purchased fromLGSS, Transferbureau Nijmegen, the Netherlands.

Phenylacetyl amide was added to a final concentration of 15 mM tominimal M63 medium supplemented with 0.2% of either succinate, glycerolor glucose as carbon source, and thiamine (1 μg/ml), L-proline (10μg/ml), and the appropriate antibiotic. All salts in the basal mediumwere replaced by the corresponding salts containing either Na⁺ or K⁺ions in order to ensure selective growth on the amide. Amides with thedesired side-chains were purchased from commercial suppliers or preparedaccording to standard techniques. E. coli strains JM101, WK6, HB101,PC2051 and PC1243 were used as hosts to select for mutant genes withspecificity for the selective amides.

Isolation and purification wild type and mutant acylases.

Cells were harvested by centrifugation and resuspended in 10 mM sodiumphosphate buffer pH 7.4 containing 140 mM NaCl. The cells were disruptedthrough sonification (6×20 sec, 100 W, 100 mm bar, Labsonic 1510: afterevery 20 seconds the cells were cooled on ice for 30 seconds).Subsequently, the suspension was centrifugated. The sonificationprocedure was repeated with the resuspended pellet and finally the celldebris was removed by centrifugation. Via ultra-filtration thesupernatant is extensively washed with milli-Q water and subsequentlywith the starting buffer for the Q-Sepharose: 20 mM NaH₂ PO₄.H₂ O pH 7.0+azide. Filter system supplied by Filtron with a Verder pump. The cutoff of the filter is 5 Kda. After ultrafiltration the sample is dilutedwith milli-Q until the conductivety is less or equal to the startingbuffer.

The sample is applied to a Q-sepharose column equilibrated with 20 mMNaH₂ PO₄.H₂ O pH 7.0+0.02% azide (conductivity=2.60 mS) and run at aflow of 20ml/min. The gradient (in 50 min to 100% 20 mM NaH₂ PO₄.H₂ O+0.5M NaCl pH 7.0+0.02% azide) was started after having washed thecolumn thoroughly with starting buffer. Detection as at 280 nm. In anext step the acylase was further purified on Hydroxylapetit(HA-ultragel IBF) equilibrated with 10 mM NaH₂ PO₄.H₂ O +10 μM CaCl₂+0.02% azide pH 6.8. The column is run at 4ml/min. The acylase elutes inequilibration buffer. The column is regenerated with 350 mMNaH2PO4.H20+10 μM CaCl₂₊₀.02 % azide pH 6.8. In case very pure proteinis required the first column step (Q-sepharose) is repeated with alonger column.

Protein concentration.

The total protein content during isolation and purification wasdetermined using the Bradford method with BSA standard. The proteinconcentration of pure A. faecalis Penicillin G acylase can be calculatedfrom the molar extinction coefficient at 280 nm. The molar extinctioncoefficient was calculated using the amino acid composition. The molarextinction coefficient calculated was 161210 M⁻¹ cm⁻¹ which correspondswith an OD of 1.87 for 1 mg/ml at a 1 cm path.

The concentration of catalytic centres of the wild type enzyme wasdetermined by titration of penicillin acylase withPhenyl-methylsulphonylfluoride (PMSF) dissolved in isopropanol atdifferent concentrations. In addition the acylase content of the finalacylase samples was determined with analytical reversed phasechromatography. Column: RP300 7micron 20×2.1 mm. Injection volume 5 μl.The protein was eluted using a linear gradient starting with 100% A(water) and changing to 80% B (70% acetonitrile in water) in 45 minutes.The acylase is eluted in two peaks corresponding to the α and β subunit.Because the acylase content of the samples which was calculated from theactive site titration experiments was found to be in line with theacylase content calculated from HPLC data, acylase mutants which did nottitrate very well with PMSF were applied to RP-HPLC in order todetermine the acylase content.

Penicillin acylase activity was assayed using NIPAB as a substrate.

Enzyme assays.

In order to determine enzymatic activity the acylases were incubatedwith substrate at room temperature in buffered solution. In caseβ-lactamase impurity was expected to be present in the enzymepreparations, 1.0 mM -lactamase inhibitor 6-bromo-penicillanic acid wasadded to the assay. The reactions were stopped by adding an excess PMSF.For some mutants which were less sensitive to PMSF inhibition, thereactions were stopped by adding 0.5 M HCl or 0.5 M acetic acid untilthe pH was between 3 and 4. When reactions were subsequently analysed byHPLC, the reactions were stopped by dilution with the correpondingelution solvent (see table 1). In addition substrates were incubatedunder assay conditions in absence of enzyme. If necessary enzyme assayswere corrected for non-enzymatic hydrolysis

The composition of the reaction mixtures was determined byhigh-performance liquid chromatography (HPLC)(table 1). Concentrationswere determined by using standards of known concentration.

                  TABLE 1                                                         ______________________________________                                        Procedures for analysis of the composition of enzyme reaction                 mixtures using high-performance liquid chromatography (HPLC).                 Reactions were stopped by diluting the reaction mixture with the              appropriate solvent which is indicated in the left column. Detection          at 214 nm. Flow 1 ml/min. SDS = Sodium dodecylsulphate.                       Sample  Column     Solvent                                                    ______________________________________                                        PenG    CP-Microspher                                                                            A: 30% acetonitrile in 0.1 M KH.sub.2 PO.sub.4             1:1 with                                                                              C18        pH 3 with 0.75 g/l SDS                                     solvent A                                                                             (Chrompack,                                                                   cat. no 28410)                                                        PenV    CP-Microspher                                                                            A: 30% acetonitrile in 0.1 M KH.sub.2 PO.sub.4             1:1 with                                                                              C18        pH 3 with 0.75 g/l SDS                                     solvent A                                                                     CefG    CP-Microspher                                                                            A: 20% acetonitrile in 0.05 M KH.sub.2 PO.sub.4            1:1 with                                                                              C18        pH 3 with 0.68 g/l SDS                                     solvent A                                                                     Ampicillin                                                                            CP-Microspher                                                                            A: 15% acetonitrile in 0.05 M KH.sub.2 PO.sub.4            1:3 with                                                                              C18        pH 3 with 0.68 g/l SDS during 6 min;                       solvent A          B:A with 50% acetonitrile during 16                                           min.                                                       PGA     CP-Microspher                                                                            A: 25% acetonitrile in 0.05 M KH.sub.2 PO.sub.4            1:1 with                                                                              C18        pH 3 with 0.68 g/l SDS                                     solvent A                                                                     Amoxicillin                                                                           Chromspher A: 25% acetonitrile in 0.012 M KH.sub.2 PO.sub.4           1:2 with                                                                              C18        pH 2.6 with 2 g/l SDS                                      solvent A                                                                             (Chrompack,                                                                   cat. no 28267)                                                        Ampicillin,                                                                           Chromspher A: 30% acetonitrile in 0.005 M KH.sub.2 PO.sub.4           PGA,    C18        pH 3.0 with 0.68 g/l SDS                                   PG, 6APA                                                                      mixtures                                                                      ______________________________________                                    

At low concentration formation of 6-APA, 7-ACA or 7-ADCA was measured bytitration with fluorescamine. Concentrations were determined bymeasuring the fluorescence at 475 n=after 390 nm excitation. In additionthe concentrations of 6-APA, 7-ACA, 7-ADCA were determined using theindicator reaction with p-dimethylamino-benzaldehyde. Formation of aSchiff base was followed at 415 nm (K. Balasingham st Al., Biochmica etBiophysica Acta 276 (1972) 250-256).

In a continuous assay Penicillin G acylase was assayedspectro-fotometrically with the chromogenic substrate NIPAB[6-nitro-3-phenylacetamido-benzoic acid]. The liberation of3-amino-6-nitrobenzoic acid was monitored by measuring the extinction at405 nm in a Kontron 610 kinetic spectrofotometer. Measuring maximalrate, the assays were performed at 25° C. using 20 mM NaH₂ PO₄.H₂ O atpH 7.5 with 20 mM NIPAB and 100 μl enzym solution (at a properdilution). Initial rate measurements were performed with varyingconcentration of NIPAB.

The kinetics of enzymatic hydrolysis of PenG, PenV, CefG were alsostudied by alkaline titration (0, OlM KOH), using a Radiometer pH-stat.All experiments were carried out in a buffer free medium.

Initial rate measurements were performed with excess substrate over theenzyme. Catalytic parameters were derived from least-squares fitting ofthe measured initial rates plotted for various substrate concentrationsaccording to the Michaelis-Menten equation.

Deacylation of the acylated L-amino acids which were used in thescreening was performed by incubation of the acyl amino acids withenzyme. Subsequently the deacylated amino acids were labeled by a methodbased on reaction with o-phthaldehyde and mercaptoethanol andquantitated using reversed phase HPLC.

Synthesis reactions were carried out in a pH-stat or in a bufferedsolution. Typical conditions used: lOmM PGA, pH 7.0, 30° C. and 30 mM6-APA. Products were analysed and quantitated by HPLC.

The reaction conditions under which the acylases were tested depend onvarious parameters, in particular the reagents, reaction time,temperature and enzyme concentration. The preferred conditions can bereadily determined by the man skilled in the art. Generally, thereaction temperature may vary between 0° and 40° C.

Examples of semi-synthetic β-lactams that may be produced by theapplication of the mutant acylase of this invention are amoxicillin,ampicillin, cefaclor, cefadroxil, cefprozil, cephalexin, and cephradine.

The acylating agens may be a derivative of D(-)-phenylglycine,D(-)-4-hydroxyphenylglycine or D(-)-2,5-dihydro-phenylglycine such as alower alkyl (methyl, ethyl, n-propyl or isopropyl) ester or an amidewhich is unsubstituted in the --CONH2 group.

Generally, the reaction temperature of the process of this invention mayvary between 0° C. and 35° C.

EXAMPLES Example 1

Exploring the environment of Penicillin G in the PenicillinG-acylase:PenG complex and identification of residue position whichaffect the catalytic properties of Penicillin G acylase.

The solvent accessible surface of the A. faecalis Penicillin G acylaseactive site was calculated using the Connolly algorithm. The probe sizewas 1.4 Å. Contouring of the accessiblity using Molecular Graphicsrevealed a deep hydrofobic cavity near the β-subunit N-terminal serinewhich was accessible from the solvent. Computer aided docking showedthat the phenylacetate fits perfectly in this cavity. After positioningthe phenylacetate in the cavity the β-subunit N-terminal serine is in anideal position for nucleophilic attack at the peptide carbonyl of PenG.

In the subsequent step the β-lactam moiety is positioned while keepingthe phenyl-acetyl group fixed in its binding pocket. Atomic overlapbetween substrate and enzyme is avoided as much as possible whilepositive interactions are maximized. Relevant positive interactionswhich contribute to binding are hydrogen bonding, electrostaticinteractions and favourable VanderWaals contacts. Hydropholicinteraction was estimated from the accessible non-polar surface which isburied by binding the substrate to the enzyme.

After manual manipulation of the substrate additional computationaltechniques were applied to optimize the substrate-enzyme complex. Energyminimization and molecular dynamics of the complex were performed usingthe CVFF forcefield (Biosym Technologies). Minimization was performed ina number of discrete steps. Minimization stopped when first derivativeenergy less than 0.01 kca1/mol

First the complexed PenG substrate was minimized while keeping theacylase atoms fixed. The distance serine B1 OG to PenG scissile carbonylcarbon was constrained between 2 and 3.5 Å.

No charges were considered.

Then hydrogen atoms of the acylase were allowed to move.

Subsequently the side chains which have at least one atom within 12 Å ofthe PenG substrate are allowed to shift while still keeping the backbonefixed. The distance serine B1 OG to PenG scissile carbonyl carbon wasstill constrained between 2 and 3.5 Å. No charges considered.

After optimization of the side chains also the main chain was allowed tomove. First movement was restricted due to tethering the main chainatoms. Gradually the tethering force was relaxed.

The initial model obtained in this way was used to analyse theenvironment of the PenG molecule. FIG. 4a shows the residues which formthe binding site of the phenylacetate moiety of the PenG substrate.Chain segments involved comprise: A139 to A152 [SEQ ID NO:27], B1, B2,B20 to B25, B31, B32, B49 to B52, B56, B57, B65 to B72, B154 to B157,B173 to B179, B239 to B241 and B476 to B480 [SEQ ID NO:32] Table 2reviews residues which have at least one atom within 8 Å from the PenGphenylacetyl moiety. This survey supplies insight in the residues whichinteract with the side chain moiety of the penicillin molecule.Essential residues for catalysis should not be replaced as substitutionleads to severely crippled or inactive acylases. These residuescomprise: B:Serl, B:Gln23, B:Asn241

Residues in A. faecalis Penicillin G acylase which are of particularinterest for binding penicillin side chain are: A:Met143, A:Phe147, [SEQID NO:27], B:Pro22, B:Phe24, B:Tyr31, B:Thr32, B:Pro49, B:Tyr52,B:Leu56, B:Phe57, B:Gly66, B:Ala67, B:Thr68, B:Ala69, B:Gly70, B:Pro71,B:Trp154, B:Val157, B:Met173, B:Ile175, B:Ser176, B:Ile177, B:Trp179[SEQ ID NO:27].

In addition the environment of the β-lactam moiety 6-APA was mapped.Table 3 reviews residues which have at least one atom within 8 Å from anatom in the PenG 6-APA moiety. FIG. 4b shows the residues which form thebinding site of the β-lactam moiety of the PenG substrate. Chainsegments involved comprise: A146 to A150 [SEQ ID NO:27] B21 to B27, B71,B250 to B263, B379 to B387, B390, B454 to B456, and B474 to B477 [SEQ IDNO:32]. FIG. 4b shows the A. faecalis Penicillin G acylase active sitefocussing on the residues around the β-lactam moiety

Residues in A. faecalis Penicillin G acylase which are of particularinterest for binding the penicillin β-lactam part are: A:Arg146,A:Phe147, A:Thr150, [SEQ ID NO:27] B:Gly25, B:Tyr27, B:Ala69, B:Pro7l,B:Thr251, B:Thr253, B:Tyr254, B:Tyr255, B:Trp256, B:Arg261, b:Met262,B:Asn379, B:Pro380, B:Gly381, B:Ser382, B:Ile383, B:Asn384, B:Met387,B:Lys390, B:Thr477, B:Glu478 [SEQ ID NO:32].

                  TABLE 2                                                         ______________________________________                                        Environment of the phenylacetyl moiety in Penicillin G Acylase                complexed with PenG                                                                   Atoms in the acylase within a certain distance                                range from PenG. Only closest atoms given. Distances                  PenG atoms                                                                            in Å. Atom indication: chain-residue number:atom                  (FIG. 3)                                                                              3-4      4-5      5-6    6-7    7-8                                   ______________________________________                                        C15     B1:OG    A147:CZ  B22:C  B2:N   A147:CZ                                       B23:O    B69:CB   B24:CD2                                                                              B21:O  B25:N                                                  B241:ND2 B68:C  B67:O  B70:N                                                                  B382:OG                                                                              B71:CG                                                                        B240:CD1                                                                      B256:CZ2                                                                      B261:CZ                                                                       B477:OG1                              O16     B1:CB    A147:CE2        B2:N   B176:O                                        B69:N    B23:O           B21:O  B177:CD1                                      B241:ND2 B68:CA          B22:CA B178:ND2                                                               B24:CE2                                                                              B239:OD1                                                               B67:O  B240:CD1                                                               B70:N  B256:CZ2                                                               B71:CG B261:CZ                                                                       B382:OG                               C17     B1:OG    B22:C    B68:CA A146:CZ                                                                              A143:SD                                       A147:CE2 B69:CB   B241:ND2                                                                             B21:O  B2:N                                          B23:O                    B25:N  B31:CE2                                       B24:CD2                  B57:CZ B56:CD2                                                                B67:O  B70:N                                                                         B177:CD1                                                                      B477:OG1                              C18     B24:CE2  A147:CZ  B57:CZ A143:SD                                                                              A146:CZ                                                B1:CB    B67:O  B21:O  B2:N                                                   B22:CB   B241:ND2                                                                             B56:CD2                                                                              B20:ND2                                                B23:O           B177:CD1                                                                             B31:CE2                                                B68:CA                 B49:CG                                                 B69:CB                 B70:N                                                                         B177:CG1                                                                      B477:OG1                              C19     A147:CG  B1:OG    A143:SD                                                                              B57:CZ A142:O                                        B24:CE2  B68:C    B22:CB B70:N  A146:C                                        B69:N             B23:O         B31:CE2                                                         B56:CD2       B49:CG                                                          B67:C         B71:CD                                                          B177:CD1      B154:CZ2                                                        B241:ND2      B176:O                                                                        B178:N                                C20     B69:N    A143:SD  B1:OG  B23:O  A142:O                                                 A147:CB  B22:CB B49:CG A146:C                                                 B24:CE2  B57:CZ B70:N  B20:ND2                                                B56:CD2  B154:CZ2                                                                             B176:O B31:CE2                                                B67:CB          B178:N B52:CE1                                                B68:C           B241:ND2                                                                             B66:C                                                  B177:CG2               B71:CD                                                                        B173:CE                                                                       B175:CG2                              C21     B24:CE2  A143:CE  A147:CD1                                                                             B20:ND2                                                                              B21:C                                         B56:CD2  B22:CB   B1:OG  B23:N  B31:CE2                                                B57:CZ   B49:CG B66:C  B52:CE1                                                B67:CB   B56:CG B178:O B70:N                                                  B68:N    B154:CZ2      B176:O                                                 B69:N    B177:CG2      B179:CD1                                                                      B241:ND2                              C22     B22:CB   B1:OG    A143:C B20:ND2                                                                              B2:O                                          B24:CE2  B56:CD2  A147:C B21:C  B32:CG2                                       B57:CZ   B67:O    B49:CG B23:O  B52:CE1                                                B68:CA          B31:CE2                                                                              B178:O                                                                 B66:C  B41:ND2                                                                B69:CB B478:OE1                                                               B154:CZ2                                                                      B177:CG2                                     C23     B1:OG    B23:N    A147:CE1                                                                             A143:CE                                                                              A146:CZ                                       B22:CB   B57:CZ   B49:CG B20:ND2                                                                              B2:N                                          B24:CD2  B67:O    B56:CD2                                                                              B21:C  B32:CG2                                                B68:CA          B31:CE2                                                                              B56:CB                                                 B69:N           B241:ND2                                                                             B154:CZ2                                                                      B177:CG2                                                                      B478:OE1                                                                      B477:OG1                              ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Environment of the 6-APA moiety in Penicillin G acylase complexed with        PenG                                                                                 Atoms in the acylase within a certain distance                                range from 6-APA moiety PenG. Only closest atoms                       PenG   given. Distances in Å. Atom indication: chain-residue              atoms  number:atom                                                            (FIG. 3)                                                                             3-4      4-5      5-6    6-7     7-8                                   ______________________________________                                        S1     A147:CE2 B23:O    A146:NE                                                                              B1:OG   B380:C                                                         B24:CA B25:N   B455:CG                                                               B69:CB                                                                        B241:OD1                                                                      B256:CZ2                                                                      B381:CA                                                                       B382:N                                        C2              A147:CE2 B381:CA                                                                              A146:NE B1:N                                                  B23:O           B24:N   B69:CB                                                                B25:N   B261:CZ                                                               B241:OD1                                                                              B379:OD1                                                              B256:NE1                                                                              B383:N                                                                B380:C  B384:ND2                                                              B382:CA                                       C3              B256:CZ2 A147:CE2                                                                             B1:N    A146:NE                                               B382:OG  B23:O  B241:CG B24:N                                                          B241:OD1                                                                             B261:CZ B25:N                                                                 B381:CA B69:CB                                                                B384:ND2                                                                              B256:CG                                                                       B379:OD1                                                                      B380:O                                                                        B383:N                                N4              A147:CE2 B23:O  B1:N    B24:N                                                 B241:OD1 B69:CB B71:CG  B381:CA                                               B256:CZ2 B382:OG                                                                              B261:CZ                                                                       B384:ND2                                      C5     A147:CE2 B69:CB   B23:O  A146:O  A150:CG2                                                       B241:OD1                                                                             B1:OG   B25:N                                                          B256:CZ2                                                                             B24:CA  B261:CZ                                                               B71:CG  B381:CA                                                               B256:CE2                                                                      B382:OG                                       C6     A147:CE2 B1:OG    B256:CZ2                                                                             B24:N   B2:N                                                  B23:O           B68:C   B22:C                                                 B69:CB          B71:CG  B70:N                                                 B241:OD1        B261:CZ B240:C                                                                B382:OGB                                                                              B241:N                                                                        B384:ND2                              C7     B241:OD1 A147:CE2 B23:O  B71:CG  B2:N                                                  B1:N     B69:CB B240:O  B24:N                                                 B256:CZ2 B261:CZ        B68:C                                                 B382:OG                 B254:CE1                                                                      B381:CA                                                                       B384:CG                               O8     B1:OD1   B23:O    A147:CE2                                                                             B69:CB  B2:N                                         B241:OD1 B256:CZ2 B240:NE1                                                                             B241:CA B22:C                                        B382:OG  B261:CZ  B382:ND2                                                                             B381:C  B24:N                                                                         B71:CG                                                                        B383:O                                                                        B390:NZ                               C9     B23:O    B381:CA  A147:CE2                                                                             A146:CZ B22:C                                        N382:N            B24:CA B1:OG   B26:CZ3                                                        B25:N  B241:OD1                                                                              B27:OH                                                         B380:O B379:OD1                                                                              B256:CZ2                                                              B383:N  B261:CZ                                                                       B384:ND2                                                                      B477:OG1                              C10                      A147:CE2                                                                             A146:CZ B256:NE1                                                       B23:O  B24:C                                                                  B380:O B25:N                                                                  B381:CA                                                                              B379:OD1                                                                      B380:C                                                                        B381:O                                                                        B382:OG                                       C11             B256:NE1 B382:OG                                                                              A147:CE2                                                                              A150:CG2                                                              B241:OD1                                                                              B23:O                                                                 B384:ND2                                                                              B69:CB                                                                        B71:CG                                                                        B261:CZ                                                                       B381:CA                               O12             B256:NE1 A147:CE2                                                                             A150:CG2                                                                              A146:O                                                                        B23:O                                                                         B69:CB                                                                        B71:CG                                                                        B241:OD1                                                                      B382:OG                                                                       B384:ND2                              O13             B256:NE1 B382:OG        A147:CE2                                                       B384:ND2       B241:OD2                                                                      B255:CD1                                                                      B256:CE3                                                                      B379:OD1                                                                      B381:CA                                                                       B383:N                                                                        B384:N                                ______________________________________                                    

Example 2

Construction of the Mutagenesis/expression Vector for Vcylase

As starting material for the construction of a combinedmutagenesis/expression vector the already described plasmid pMcTAFNdewas used (EP-453048). This vector, which was constructed from pMcTNdeand pAF1, harbors the complete penicillin acylase gene from Alcaligenesfaecalis . In order to facilitate the construction of convenient gappedduplex molecules and to facilitate the exchange of PCR overlap extensionfragments three new unique restriction sites were inserted withoutaltering the coding information: EcoRV (position 5239), Nsi1 (pas. 5972)and Cla1 (pos. 6420). The resulting vector, pMcAF, which is shown inFIG. 5, was used to construct mutant acylase genes. The mutant acylaseswere produced in E. coli WK6 or HB101 laqI^(q) under guidance of the tacpromoter provided.

Example 3

Mutagenesis of A. faecalis acylase

At selected positions amino acid mutations were generated using lo thePCR overlap extension method described before. The amino acid positionsin the respective subunit (A or B) are shown in table 4. Theoligonucleotides used for the construction are also shown. Note that atposition A143 and B67, B68, B69 an oligo with randomized codons wasused.

Example 4

Assay of site directed mutants of penicillin acylase for correct foldingand post translational processing using suitable auxotrophs of E. coli

E. coli HB101 laqI^(q) cells harbouring the identified mutant acylasegenes were tested on agar plates containing selective media.

Selective media for phenylacetyl L-leucine (`fal` ) were prepared asdescribed by Garcia (supra). Minimal plates are as follows: M9 minimalagar, 1 mg/l thiamine, 10 mg/l L-proline, 0.2 mM IPTG and theappropriate antibiotic (50 μg/ml chloramphenicol (cap) or 75 μg/mlampicillin (amp)). The available data from literature on expression ofpenicillin acylase indicate that proper folding and posttranslationalprocessing of the chain are critical factors for obtaining catalyticalviable penicillin acylase. In order to establish whether the mutantpenicillin acylase is expressed properly as an active acylase 200 μg/mlof an acyl L-leucine was included into minimal plates. Transformants ormutants of E. coli HB101 (Leu) growing exclusively in the presence ofthe phenyl-acetyl-L-leucine are considered to harbour an active properlyexpressed penicillin acylase gene. Table 5 shows the result for severalselected mutants.

In addition this method may be employed for an inital rough screeningfor acylases with an altered specificity. For selections on side-chainspecificity of acylases 200 μg/ml of a desired acyl L-leucine wasincluded into minimal plates. In case the acyl moiety is not recognizedby the wild type penicillin acylase transformants or mutants of E. coliHB101 (Leu) growing exclusively in the presence of the desired acylL-leucine are considered to harbour an acylase gene with the desiredspecificity (e.g. glutaryl-L-leucine). Examples of such selectivesubstrates are a-D-aminoadipyl leucine, adipyl-leucine and glutarylleucine. These compounds were purchased from LGSS, TransferbureauNijmegen, The Netherlands.

                                      TABLE 4                                     __________________________________________________________________________    Synthetic DNA-oligonucleotides for PCR mutation.                              (X = all possible amino acids)                                                (R = A or G; Y = C or T; S = C or G; W = A or T; B = C, G or T;                V = A, C, G; N = A, C, G or T)                                               A.A.-                                                                              A.A.-                                                                    position                                                                           mutation                                                                           DNA-oligonucleotides: 5'-3'                                         __________________________________________________________________________    A143 M: R, K                                                                            5' GGGTGGGCTCCARGGCCAATCG 3'    [SEQ ID NO:1]                                 5' GCGATTGGCCYTGGAGCCCAC 3'     [SEQ ID NO:2]                       A147 F: Y, h                                                                            5' TGGGCTCCATGGCCAATCGCYACTCCGACACGAA 3'                                                                      [SEQ ID NO:3]                            F: W 5' TGGGCTCCATGGCCAATCGCTGGTCCGACACGAA 3'                                                                      [SEQ ID NO:4]                       B24  F:R, K                                                                             5' CGGCCCACAGARGGGCTGGTACA '    [SEQ ID NO:5]                                 5' GTACCAGCCCYTCTGTGGGCC 3'     [SEQ ID NO:6]                       B56  L: R, K                                                                            5' TCCGATCGTAARGTTTGGCACC 3'    [SEQ ID NO:7]                                 5' GGTGCCAAACYTTACGATCGGAT 3'   [SEQ ID NO:8]                            L: H 5' TCCGATCGTACATTTTGGCACC 3'    [SEQ ID NO:9]                                 5' GGTGCCAAAATGTACGATCGGAT 3'   [SEQ ID NO:10]                           L: G, A, V                                                                         5' CCGATCGTAGBCTTTGGCAC 3'      [SEQ ID NO:11]                                5' GTGCCAAAGVCTACGATCGG 3'      [SEQ ID NO:12]                      B71  P: F, Y                                                                            5' GCTGGCTWCCAAGATGTGGTG 3'     [SEQ ID NO:13]                                5' ATCTTGGWAGCCAGCAGTCGC 3'     [SEQ ID NO:14]                      B177 I:R, K                                                                             5' GATGGCGATATCCARGAACTGGTACTA 3'                                                                             [SEQ ID NO:15]                           I: H 5' GATGGCGATATCCCACAACTGGTACTA 3'                                                                             [SEQ ID NO:16]                           I: C, M                                                                            5' CAGCAAGATGGCGATATCCRTGAACTGGTACTACGC 3'                                                                    [SEQ ID NO:17]                           I: S, T                                                                            5' CAGCAAGATGGCGATATCCASCAACTGGTACTACGC 3'                                                                    [SEQ ID NO:18]                      A143 M: X 5' GGGTGGGCTCCNNSGCCAATCGCTTCTC 3'                                                                            [SEQ ID NO:19]                                5' AAGCGATTGGCSNNGGAGCCCACCCAG 3'                                                                             [SEQ ID NO:20]                      B67  A: S, G, T                                                                         5' GGGR/SCACTGCTGGGCCTCAAG 3'   [SEQ ID NO:21]                                5' AGTGS/YCCCCAGGCAATCTC 3'     [SEQ ID NO:22]                           A: S, G                                                                            5' GCCTGGGGGRCACTGCTGGCCCGCAAG 3'                                                                             [SEQ ID NO:23]                                5' GCCAGCAGTGCYCCCCCAGGCAATCTC 3'                                                                             [SEQ ID NO:24]                      B67  A: X 5' CGAGATTGCCTGGGGGNNSNNSNNSGGCCCGCAAGATGTGGTGGAC 3'                                                          [SEQ ID NO:25]                      B68  T: X 5' CCACATCTTGCGGGCCSNNSNNSNNCCCCCAGGCAATCTCGC 3'                                                              [SEQ ID NO:26]                      B69  A: X                                                                     __________________________________________________________________________

When wild type has low activity for an acyl group, mutants withincreased activety can be picked up with this method by comparing thesize of the halo produced by the mutant with respect to wild type.Useful side chains are phenoxyacetyl, p-hydroxyphenylglycyl,phenylglycyl.

                  TABLE 5                                                         ______________________________________                                        In vivo specifity of mutant acylsases. A and B in the first column            refers to α and β subunit. ++ growth rate comparable to wild       type;                                                                         + growth rate reduce with respect to wild type; - no growth during            3 weeks.                                                                                          fenyl-acetyl-                                                    mutant       L-leucine                                                 ______________________________________                                               A:M143R      +                                                                A:M143K      +                                                                A:F147Y      ++                                                               A:F147H      ++                                                               A:F147W      ++                                                               B:F24R       -                                                                B:F24K       ++                                                               B:L56R                                                                        B:L56K       ++                                                               B:L56H                                                                        B:I177R                                                                       B:I177K      ++                                                               B:I177H      ++                                                        ______________________________________                                    

Instead of leucine also the amino acid moiety of the selective substratecan be varied. In such case a suitable auxotrophic mutant of E. coli wasused for selection. Instead also amide of the acyl moiety are usefulcomponds for selection. Side-chain amide (e.g. phanylacetylamide,glutarylamide, adipylamide, α-aminoadipylamide) was added to a finalconcentration of 15 mM to minimal M9 medium supplemented with 0.2% ofeither succinate, glycerol or glucose as carbon source, and thiamine (1μmg/ml), L-proline (10 μg/ml), 0.2 mM IPTG and the appropriateantibiotic.

All ammonium salts in the basal medium were replaced by thecorresponding salts containing either Na⁺ or K⁺ ions in order to ensureselective growth on the amide. Amides with the desired side-chains werepurchased from commercial suppliers or prepared according to standardtechniques. E. coli strains JM101, WK6 and HB101 were used as hosts toselect for mutant genes with specificity for the selective amides.

Example 5

Assay on targeted random mutants of Penicillin Acylase

In case of TRM mutagenesis a pool of mutants was plated on selectiveplates prior to DNA sequencing. Only the colonies which showed growth onone ore more of the selective media were characterized. The result for 2TRM mutagenesis experiments are shown in table 6.

                  TABLE 6                                                         ______________________________________                                        In vivo specificity of mutant acylases. A in the first column refers          to the α subunit. ++ growth rate comparable to wild type; + growth      rate reduce with respect to wild type; - no growth during 3 weeks.            mutant            fenyl-acetyl-l-leucine                                      ______________________________________                                        A:M143C           ++                                                          A:M143G           +                                                           A:M143D           +                                                           A:M143T           ++                                                          A:M143V           ++                                                          A:M143L           ++                                                          ______________________________________                                    

Example 6

Increased Specific Activity and Altered Specificity

The catalytic parameters of A.faecalis PenG acylase mutants weredetermined for different substrates. The altered specificities for themutants are exemplified in Tables 7 and 8. Compared to wild type themutants A:M143V and B:L56K exhibit a higher turn-over rate for thedeacylation of PenV and CefG. A:F147Y is more active compared to wildtype when used in the deamidation of D-phenylglycinamide.

At high substrate concentrations, which is usually the situation in manyindustrial conversion processes, the acylase will be completelysaturated with substrate and as a consequence the conversion willproceed at maximal velocity. In FIG. 6 the maximal velocity for a numberof substrates is plotted for the wild type A. faecalis acylase and forsome mutants. Velocities are scaled relative to PenG whereby V_(sax) forPenG has been set to 1. Wild type PenG acylase shows the highestactivity for PenG as was expected. However the substitution A:M143Vturns the enzyme into a CefG acylase, while the substitution A:F147Yturns the enzyme into a powerful amidase for the deamidation ofD-Phenylglycinamide ((D)PGA). In addition the deacylation velocities ofA:F147Y are higher for ampicillin and NIPAB than for PenG. In FIG. 7 theV_(sax) value which was measured for mutants B:L56G, B:L56A, B: L56V,B:177V, BI177S, B:A67S, B:A67G for the given substrates is compared tothe V_(sax) for PenG in a similar way as was done in FIG. 6. Specificityhas shifted with respect to wild type. E.g. mutant B:1l77S exhibits areduced deacylation rate on ampicilin and an improved activiry onD-phenylglycinamide ((D)PGA).

In general the specificity or selectivity of an enzyme in the sense ofdiscrimination between two competing substrates is determined bycomparing the V_(sax) /K_(s) (or k_(cat) /K_(s)) of the two substrates.In table 9 this ratio has been compared for different substratecombinations. Especially the considerable increase of the specificity ofthe A:F147Y mutant for (D)PGA is striking.

                  TABLE 9                                                         ______________________________________                                        Selectivity of the wild type enzyme compared to selectivity                   of the mutants for a number of substrates.                                    (V.sub.max /K.sub.s).sub.s1 /                                                 (V.sub.max /K.sub.s).sub.s2                                                   (×100) wild                                                             S1       S2      type   A:M143V  B:L56K                                                                              A:F147Y                                ______________________________________                                        PenV     PenG    0.97   4.93     1.21  1.10                                   CefG     PenG    54.95  119.33   7.00  44.44                                  PenV     CefG    1.76   4.13     17.35 2.48                                   Ampi     PenG    14.80  22.70    3.40  35.30                                  (D)PGA   Ampi    9.50   4.10     3.00  367.60                                 ______________________________________                                    

                                      TABLE 7                                     __________________________________________________________________________    Catalytic parameters K.sub.m and V.sub.max as were determined for wild        type Alcaligenes faecalis                                                     PenG acylase and some mutants. Assay conditions: NIPAB, 0.1M                  NaH2PO.sub.4,                                                                 pH 7.5, 25° C.; PenG and PenV, 40 mM NaH.sub.2 PO.sub.4, pH 7.5,       37° C.;                                                                CefG, 20 mM NaH.sub.2 PO.sub.4, pH 7.5, 37° C.; Amoxi(cillin),         Ampi(cillin) and D-Phenylglycineamide                                         ((D)PGA), 20 mM NaH.sub.2 PO.sub.4, pH 7.0, 37° C.                     wild type       A:M143V    B:L56K     A:F147Y                                            V.sub.max  V.sub.max  V.sub.max  V.sub.max                         K.sub.m (μM)                                                                          (U/mg)                                                                             K.sub.m (μM)                                                                     (U/mg)                                                                             K.sub.m (μM)                                                                     (U/mg)                                                                             K.sub.m (μM)                                                                     (U/mg)                            __________________________________________________________________________    NIPAB                                                                              4     37.0 17    35.7 28    47.3 5     18.0                              PenG 2     45.5 6     40.5 1     36.4 4     5.9                               PenV 18    4.0  31    10.3 35    15.5 51    0.8                               CefG 1     12.5 7     56.4 10    25.5 6     4.0                               Ampi 700   23.5 1500  23.0 1700  20.9 2600  13.6                              (D)PGA                                                                             8600  27.5 35000 22.2 49000 18.2 1700  32.7                              Amoxi                                                                              14000 .9   21000 .2              19000 0.1                               __________________________________________________________________________

                  TABLE 8                                                         ______________________________________                                        Catalytic parameters K.sub.s and V.sub.max as were determined for wild        type                                                                          Alcaligenes fascalis PenG acylase and some mutants. Assay condi-              tions: NIPAB, 0.1 M NaH2PO.sub.4, pH 7.5, 25° C. For mutants           B:A678 and                                                                    B:A67G V.sub.max in U/ml.                                                              K.sub.s (μM)                                                                            V.sub.max (U/mg)                                                                        V.sub.max /K.sub.s                            ______________________________________                                        wt AF    4            37.0      9.3                                           B:L56G   12           21.3      1.8                                           B:L56A   14           37.1      2.7                                           B:L56V   9            28.2      3.1                                           B:I177V  10           34.5      3.5                                           B:I177S  76           30.2      0.4                                           B:A67S   5            7.1                                                     B:A67G   11           1.1                                                     ______________________________________                                    

Example 7

Improved Stereospecificity of PenG Acylase.

Wild type A.faecalis and E. coli PenG acylase show a preference for theD enantiomer of penicillins with an α-carbon substituted side chain.Examples are ampicillin, cefalexin, amoxicillin, cefadroxyl, andcefaclor. An increased stereospecifity of Penicillin G acylases isdesired in order to obtain Penicillin G acylase which shows an improvedenantiomeric excess in conversions with racemic mixtures of chiralcompounds. Such property makes the Penicillin G acylase extremely usefulfor synthesis of enantiomerically pure semisynthetic antibiotics fromracemic mixtures of α-carbon substituted phenylacetyl side chains oractivated derivatives of the α-carbon substituted phenylacetyl sidechain (e.g. phenylglycine-amides or -esters,p-hydroxyphenylglycine-amides or -esters) which contain a chiralα-carbon due to the presence of an amino group (e.g. Ampicillin,Cefalexin, Amoxycillin, Cefadroxyl, Cephachlor) or a hydroxyl group(Cephamandol).

Table 10 shows that for phenylglycinamide wild type PenG acylases show apreference for the D enantiomer. For a racemic mixture (1:1) of D and Lphenylglycineamide v_(D/v) _(L) equals (V_(sax) /K_(s))^(D-PGA)/(V_(sax) /K_(s))^(L-PGA) where v_(D) and v_(L) represent velocities ofdeamidation of D and L enantiomer respectively. So for the wild type A.faecalis the velocity of deamidation of the D enantiomer is 5 timesfaster than for the L enantiomer. For mutant A:F143Y thesteroselectivity which is expressed as (V_(sax) /K_(s))^(D-PGA)/(V_(sax) /K_(s))L^(-PGA) has increased from 5.10 to 36.52. This meansthat the velocity of deamidation of D enantiomer is 36.52 times fasterthan that of L instead of only 5.10 times as for the wild type.

                  TABLE 10                                                        ______________________________________                                        Stereospecificity of the wild type enzymes A. faecalis and E. coli            versus stereospecificity of the mutants for DL-phenylglycinamide              (PGA). Assay conditions DL phenylglycineanide (PGA): 20 mM                    NaH.sub.2 PO.sub.4, pH 7.0, 37° C.                                                   (V.sub.max /K.sub.s).sup.D-PGA /(V.sub.max /K.sub.s).sup.L-P                  GA                                                              ______________________________________                                        Wild type E. coli                                                                           3.32                                                            Wild type A. faecalis                                                                       5.10                                                            A:M143V       5.70                                                            B:L56K        3.25                                                            A:F147Y       36.52                                                           ______________________________________                                    

Example 8

Reduced product Inhibition.

The complete conversion of NIPAB was followed as a funtion of time at20, 50 and 100 μM NIPAB by following the increase in absorbtion at 405nm. Products of this conversion are phenylacetic acid and3-amino-6-nitrobenzoic acid. The conversion was performed at 25° C. in0.1 M NaH₂ PO₄.H₂ O buffer pH 7.5. The progress curves of thedeacylation of NIPAB could be fitted very well when product inhibitionby phenylacetic acid was taken into account. The dissociation constants(usually referred to as inhibition constant K_(i)) for phenylacetic acidwhich could be derived from the progress curves is shown in table 11.The benefits of some mutants which are less sensitive to productinhibition are shown in table 12. For these mutants the yield of theconversion in a fixed time span is higher than for wild type.Alternatively, in order to obtain a certain yield a shorter conversiontime is needed for the mutants.

The conversion of PenG is usually performed at concentrations as high as200 mM. Using an identical amount of PenG units, the mutant A:M143V mayreach in 20 minutes a conversion yield of 90% while wild type approaches84% in this time span.

                  TABLE 11                                                        ______________________________________                                        Inhibition of PenG acylase by phenylacetic acid (PA). K.sub.i                 (inhibition                                                                   constant PA) represents the dissociation constant. The catalytic              parameters were determined at 25° C. in 0.1 M NaH.sub.2 PO.sub.4.H.    sub.2 O buffer pH                                                             7.5.                                                                                       K.sub.i.sup.Phenylacetic acid (μM)                            ______________________________________                                        wt AF        11                                                               B:L56K       115                                                              B:L56V       31                                                               B:L56A       59                                                               B:L56G       55                                                               B:A67G       65                                                               B:I177S      252                                                              B:I177V      35                                                               A:M143V      74                                                               ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        Progress of the NIPAB conversion in time. The yield represents the            fraction of substrate which has been converted. Conversion of 200 μM       NIPAB, 25° C. in 0.1 M NaH.sub.2 PO.sub.4.H.sub.2 O buffer pH 7.5      using 0.1 Unit of                                                             enzyme (NIPAB units).                                                                        Yield (%)                                                                              Yield (%)                                                            15 min   30 min                                                ______________________________________                                        wt AF          61.8     91.8                                                  B:L56A         61.3     93.3                                                  B:L56G         62.3     94.5                                                  B:A67G         63.8     96.4                                                  A:M143V        60.5     92.7                                                  ______________________________________                                    

Example 9

Altered molar ratio aminolysis/hydrolysis. The synthesis of ampicillinfrom (D)phenylglycinamide(D-PGA) and 6APA using PenG acylases. To abuffered solution containing (D)phenylglycinamide(D-PGA) and 6APA PenGacylase wild type or mutants were added. At different time intervalssamples were analyzed and the composition of the samples was determinedaccording to the methods described in the experimental section. Theresults are shown in tables 13 and 14. Some mutants show improved molarratio aminolysis/hydrolysis.

                  TABLE 13                                                        ______________________________________                                        Molar ratio aminolysis or synthesis versus hydrolysis (S/H) obtained          in the synthesis of ampicillin by PenG acylases. Initial                      concentrations 12.4 mM D-PGA and 62 mM ampicillin. Experimental               conditions: 0.1 M Tris buffer pH 7.8, temperature 4° C., enzymes       dosed                                                                         at 0.7 NIPAB units per ml.                                                              Aminolysis/Hydrolysis molar ratio:                                            Ampicillin/D-Phenylglycine                                                    t = 5 min                                                                              t = 15 min t = 30 min                                                                           t = 60 min                               ______________________________________                                        wt AF     0.95     0.92       0.69   0.36                                     A:M143V   0.75     0.92       0.94   0.71                                     B:L56G    0.78     1.03       1.02   0.79                                     B:I177S   0.30     0.79       1.05   1.17                                     ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Molar ratio aminolysis or synthesis over hydrolysis (S/H) obtained            in the synthesis of ampicillin by PenG acylases. Initial                      concentrations 10 mM D-PGA and 30 mM ampicillin. Experimental                 condition: 0.1 M Tris buffer pH 7.8, temperature 25° C., enzymes       dosed                                                                         at 1.4 D-PGA units per ml.                                                             Aminolysis/Hydrolysis molar ratio:                                            Ampicillin/D-Phenylglycine                                                    t = 10 min    t = 30 min                                                                             t = 60 min                                    ______________________________________                                        wt AF    0.43          0.20     0.06                                          A: M143V 0.50          0.31     0.15                                          ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 36                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #                 22AAT CG                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #21                CCCA C                                                     - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #        34        TCGC YACTCCGACA CGAA                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #        34        TCGC TGGTCCGACA CGAA                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #                23TGGT ACA                                                   - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #21                GGGC C                                                     - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #                 22GCA CC                                                    - (2) INFORMATION FOR SEQ ID NO:8:                                            - 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     (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                # 20               GCAC                                                       - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                # 20               TCGG                                                       - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #21                TGGT G                                                     - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #21                GTCG C                                                     - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #             27   AACT GGTACTA                                               - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #             27   AACT GGTACTA                                               - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #       36         TCCR TGAACTGGTA CTACGC                                     - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #       36         TCCA SCAACTGGTA CTACGC                                     - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #             28   CAAT CGCTTCTC                                              - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #             27   AGCC CACCCAG                                               - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #21                TCAA G                                                     - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                # 20               TCTC                                                       - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #             27   TGGC CCGCAAG                                               - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #             27   CAGG CAATCTC                                               - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #                 46NSN NSNNSGGCCC GCAAGATGTG GTGGAC                          - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #  42              SNNS NNSNNCCCCC AGGCAATCTC GC                              - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 202 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Gln Val Gln Ser Val Glu Val Met Arg Asp Se - #r Tyr Gly Val Pro His         #                15                                                           - Val Phe Ala Asp Ser His Tyr Gly Leu Tyr Ty - #r Gly Tyr Gly Tyr Ala         #            30                                                               - Val Ala Gln Asp Arg Leu Phe Gln Met Asp Me - #t Ala Arg Arg Ser Phe         #        45                                                                   - Val Gly Thr Thr Ala Ala Val Leu Gly Pro Gl - #y Glu Gln Asp Ala Tyr         #    60                                                                       - Val Lys Tyr Asp Met Gln Val Arg Gln Asn Ph - #e Thr Pro Ala Ser Ile         #80                                                                           - Gln Arg Gln Ile Ala Ala Leu Ser Lys Asp Gl - #u Arg Asp Ile Phe Arg         #                95                                                           - Gly Tyr Ala Asp Gly Tyr Asn Ala Tyr Leu Gl - #u Gln Val Arg Arg Arg         #           110                                                               - Pro Glu Leu Leu Pro Lys Glu Tyr Val Asp Ph - #e Asp Phe Gln Pro Glu         #       125                                                                   - Pro Leu Thr Asp Phe Asp Val Val Met Ile Tr - #p Val Gly Ser Met Ala         #   140                                                                       - Asn Arg Phe Ser Asp Thr Asn Leu Glu Val Th - #r Ala Leu Ala Met Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Ser Leu Glu Lys Gln His Gly Pro Glu Ar - #g Gly Arg Ala Leu Phe         #               175                                                           - Asp Glu Leu Leu Trp Ile Asn Asp Thr Thr Al - #a Pro Thr Thr Val Pro         #           190                                                               - Ala Pro Ala Ala Glu His Lys Pro Gln Ala                                     #       200                                                                   - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 209 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Ar - #g Asp Glu Tyr Gly Met         #                15                                                           - Pro His Ile Tyr Ala Asn Asp Thr Trp His Le - #u Phe Tyr Gly Tyr Gly         #            30                                                               - Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Me - #t Glu Met Ala Arg Arg         #        45                                                                   - Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gl - #y Lys Asp Phe Val Lys         #    60                                                                       - Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pr - #o Asp Ala Ile Arg Ala         #80                                                                           - Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Se - #r Ile Leu Gln Gly Tyr         #                95                                                           - Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Va - #l Asn Thr Asn Pro Glu         #           110                                                               - Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gl - #y Phe Thr Pro Lys Arg         #       125                                                                   - Trp Glu Pro Phe Asp Val Ala Met Ile Phe Va - #l Gly Thr Met Ala Asn         #   140                                                                       - Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp As - #n Leu Ala Leu Leu Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gl - #y Met Ala Val Phe Asn         #               175                                                           - Gln Leu Lys Trp Leu Val Asn Pro Ser Ala Pr - #o Thr Thr Ile Ala Val         #           190                                                               - Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gl - #n Gln Asn Ser Gln Thr         #       205                                                                   - Ala                                                                         - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 209 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Ala Ser Pro Pro Thr Glu Val Lys Ile Val Ar - #g Asp Glu Tyr Gly Met         #                15                                                           - Pro His Ile Tyr Ala Asp Asp Thr Tyr Arg Le - #u Phe Tyr Gly Tyr Gly         #            30                                                               - Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Me - #t Glu Met Ala Arg Arg         #        45                                                                   - Ser Thr Gln Gly Thr Val Ser Glu Val Leu Gl - #y Lys Ala Phe Val Ser         #    60                                                                       - Phe Asp Lys Asp Ile Arg Gln Asn Tyr Trp Pr - #o Asp Ser Ile Arg Ala         #80                                                                           - Gln Ile Ala Ser Leu Ser Ala Glu Asp Lys Se - #r Ile Leu Gln Gly Tyr         #                95                                                           - Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Va - #l Asn Ala Ser Pro Asp         #           110                                                               - Lys Leu Leu Pro Gln Gln Phe Ser Thr Phe Gl - #y Phe Lys Pro Lys His         #       125                                                                   - Trp Glu Pro Phe Asp Val Ala Met Ile Phe Va - #l Gly Thr Met Ala Asn         #   140                                                                       - Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp As - #n Leu Ala Leu Leu Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Val Lys Asp Lys Tyr Gly Asn Asp Glu Gl - #y Met Ala Val Phe Asn         #               175                                                           - Gln Leu Lys Trp Leu Val Asn Pro Ser Ala Pr - #o Thr Thr Ile Ala Ala         #           190                                                               - Arg Glu Ser Ser Tyr Pro Leu Lys Phe Asp Le - #u Gln Asn Thr Gln Thr         #       205                                                                   - Ala                                                                         - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 207 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Ala Lys Asn Glu Gly Val Lys Val Val Arg As - #p Asn Phe Gly Val Pro         #                15                                                           - His Leu Tyr Ala Lys Asn Lys Lys Asp Leu Ty - #r Glu Ala Tyr Gly Tyr         #            30                                                               - Val Met Ala Lys Asp Arg Leu Phe Gln Leu Gl - #u Met Phe Arg Arg Gly         #        45                                                                   - Asn Glu Gly Thr Val Ser Glu Ile Phe Gly Gl - #u Asp Tyr Leu Ser Lys         #    60                                                                       - Asp Glu Gln Ser Arg Arg Asp Gly Tyr Ser As - #n Lys Glu Ile Lys Lys         #80                                                                           - Met Ile Asp Gly Leu Asp Arg Gln Pro Arg Gl - #u Leu Ile Ala Lys Phe         #                95                                                           - Ala Glu Gly Ile Ser Arg Tyr Val Asn Glu Al - #a Leu Lys Asp Pro Asp         #           110                                                               - Asp Lys Leu Ser Lys Glu Phe His Glu Tyr Gl - #n Phe Leu Pro Gln Lys         #       125                                                                   - Trp Thr Ser Thr Asp Val Val Arg Val Tyr Me - #t Val Ser Met Thr Tyr         #   140                                                                       - Leu Trp Ile Ile Thr Arg Glu Leu Lys Asn Al - #a Glu Ile Leu Ala Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Glu His Glu Tyr Gly Thr Glu Val Ser Ar - #g Lys Met Phe Asp Asp         #               175                                                           - Leu Val Trp Lys Asn Asp Pro Ser Ala Pro Th - #r Ser Ile Val Ser Glu         #           190                                                               - Gly Lys Pro Lys Arg Glu Ser Ser Ser Gln Se - #r Leu Gln Lys Leu             #       205                                                                   - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 284 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Met Lys Lys His Leu Ile Ser Ile Ala Ile Va - #l Leu Ser Leu Ser Ser         #                15                                                           - Leu Ser Leu Ser Ser Phe Ser Gln Ser Thr Gl - #n Ile Lys Ile Glu Arg         #            30                                                               - Asp Asn Tyr Gly Val Pro His Ile Tyr Ala As - #n Asp Thr Tyr Ser Leu         #        45                                                                   - Phe Tyr Gly Tyr Gly Tyr Ala Val Ala Gln As - #p Arg Leu Phe Gln Met         #    60                                                                       - Glu Met Ala Lys Arg Ser Thr Gln Gly Thr Va - #l Ser Glu Val Phe Gly         #80                                                                           - Lys Asp Tyr Ile Ser Phe Asp Lys Glu Ile Ar - #g Asn Asn Tyr Trp Pro         #                95                                                           - Asp Ser Ile His Lys Gln Ile Asn Gln Leu Pr - #o Ser Gln Glu Gln Asp         #           110                                                               - Ile Leu Arg Gly Tyr Ala Asp Gly Met Asn Al - #a Trp Ile Lys Gln Ile         #       125                                                                   - Asn Thr Lys Pro Asp Asp Leu Met Pro Lys Gl - #n Phe Ile Asp Tyr Asp         #   140                                                                       - Phe Leu Pro Ser Gln Trp Thr Ser Phe Asp Va - #l Ala Met Ile Met Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Thr Met Ala Asn Arg Phe Ser Asp Met As - #n Ser Glu Ile Asp Asn         #               175                                                           - Leu Ala Leu Leu Thr Ala Leu Lys Asp Lys Ty - #r Gly Glu Gln Leu Gly         #           190                                                               - Val Glu Phe Phe Asn Gln Ile Asn Trp Leu As - #n Asn Pro Asn Ala Pro         #       205                                                                   - Thr Thr Ile Ser Ser Glu Glu Phe Thr Tyr Se - #r Asp Ser Gln Lys Thr         #   220                                                                       - Lys Asn Ile Ser Gln Leu Asn Gln Ile Ser As - #p Tyr Arg Leu Thr Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Met Phe Glu Arg Thr Ala Lys Asp Thr Th - #r Gly Lys Val Leu Ala         #               255                                                           - Leu Ser Ser Gln Glu Asn Asn Ala Leu Ile Al - #a Lys Gln Tyr Glu Gln         #           270                                                               - Ser Gly Ala Asn Gly Leu Ala Gly Tyr Pro Th - #r Thr                         #       280                                                                   - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 551 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - Ser Asn Leu Trp Ser Thr Arg Pro Glu Arg Va - #l Gln Glu Gly Ser Thr         #                15                                                           - Val Leu Ile Asn Gly Pro Gln Phe Gly Trp Ty - #r Asn Pro Ala Tyr Thr         #            30                                                               - Tyr Gly Ile Gly Leu His Gly Ala Gly Phe As - #p Val Val Gly Asn Thr         #        45                                                                   - Pro Phe Ala Tyr Pro Ile Val Leu Phe Gly Th - #r Asn Ser Glu Ile Ala         #    60                                                                       - Trp Gly Ala Thr Ala Gly Pro Gln Asp Val Va - #l Asp Ile Tyr Gln Glu         #80                                                                           - Lys Leu Asn Pro Ser Arg Ala Asp Gln Tyr Tr - #p Phe Asn Asn Ala Trp         #                95                                                           - Arg Thr Met Glu Gln Arg Lys Glu Arg Ile Gl - #n Val Arg Gly Gln Ala         #           110                                                               - Asp Arg Glu Met Thr Ile Trp Arg Thr Val Hi - #s Gly Pro Val Met Gln         #       125                                                                   - Phe Asp Tyr Asp Gln Gly Ala Ala Tyr Ser Ly - #s Lys Arg Ser Trp Asp         #   140                                                                       - Gly Tyr Glu Val Gln Ser Leu Leu Ala Trp Le - #u Asn Val Ala Lys Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Arg Asn Trp Thr Glu Phe Leu Asp Gln Ala Se - #r Lys Met Ala Ile Ser         #               175                                                           - Ile Asn Trp Tyr Tyr Ala Asp Lys His Gly As - #n Ile Gly Tyr Val Ser         #           190                                                               - Pro Ala Phe Leu Pro Gln Arg Pro Ala Asp Gl - #n Asp Ile Arg Val Pro         #       205                                                                   - Ala Lys Gly Asp Gly Ser Met Glu Trp Leu Gl - #y Ile Lys Ser Phe Asp         #   220                                                                       - Ala Ile Pro Lys Ala Tyr Asn Pro Pro Gln Gl - #y Tyr Leu Val Asn Trp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Asn Lys Pro Ala Pro Asp Lys Thr Asn Th - #r Asp Thr Tyr Tyr Trp         #               255                                                           - Thr Tyr Gly Asp Arg Met Asn Glu Leu Val Se - #r Gln Tyr Gln Gln Lys         #           270                                                               - Asp Leu Phe Ser Val Gln Glu Ile Trp Glu Ph - #e Asn Gln Lys Ala Ser         #       285                                                                   - Tyr Ser Asp Val Asn Trp Arg Tyr Phe Arg Pr - #o His Leu Glu Lys Leu         #   300                                                                       - Ala Gln Gln Leu Pro Ala Asp Asp Ser Ser Ly - #s Ala Ala Leu Thr Met         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Leu Ala Trp Asp Gly Met Glu Gln Asp Gl - #n Gly Gly Gln Asn Ala         #               335                                                           - Gly Pro Ala Arg Val Leu Phe Lys Thr Trp Le - #u Glu Glu Met Tyr Lys         #           350                                                               - Gln Val Leu Met Pro Val Val Pro Glu Ser Hi - #s Arg Ala Met Tyr Ser         #       365                                                                   - Gln Thr Gly Phe Ala Thr Gln Gln Gly Pro As - #n Pro Gly Ser Ile Asn         #   380                                                                       - Leu Ser Met Gly Thr Lys Val Leu Leu Arg Al - #a Leu Val Leu Glu Ala         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - His Pro Asp Pro Lys Arg Val Asn Val Phe Gl - #y Glu Arg Ser Ser Gln         #               415                                                           - Glu Ile Met His Thr Ala Leu Gln Asn Ala Gl - #n Ala Arg Leu Ser Gln         #           430                                                               - Glu Gln Gly Ala Gln Met Ala Arg Trp Thr Me - #t Pro Thr Ser Val His         #       445                                                                   - Arg Phe Ser Asp Lys Asn Phe Thr Gly Thr Pr - #o Gln Thr Met Pro Gly         #   460                                                                       - Asn Thr Phe Ala Phe Thr Gly Tyr Gln Asn Ar - #g Gly Thr Glu Asn Asn         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Arg Val Val Phe Asp Ala Lys Gly Val Glu Ph - #e Cys Asp Ala Met Pro         #               495                                                           - Pro Gly Gln Ser Gly Phe Thr Asp Arg Asn Gl - #y Val Arg Ser Pro His         #           510                                                               - Tyr Glu Asp Gln Leu Lys Leu Tyr Glu Asn Ph - #e Glu Cys Lys Thr Met         #       525                                                                   - Asp Val Thr His Ala Asp Ile Arg Arg Asn Al - #a Gln Ser Ser Thr Met         #   540                                                                       - Leu Leu Ile Gln Pro Gln Pro                                                 545                 5 - #50                                                   - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 557 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - Ser Asn Met Trp Val Ile Gly Lys Ser Lys Al - #a Gln Asp Ala Lys Ala         #                15                                                           - Ile Met Val Asn Gly Pro Gln Phe Gly Trp Ty - #r Ala Pro Ala Tyr Thr         #            30                                                               - Tyr Gly Ile Gly Leu His Gly Ala Gly Tyr As - #p Val Thr Gly Asn Thr         #        45                                                                   - Pro Phe Ala Tyr Pro Gly Leu Gly Phe Gly Hi - #s Asn Gly Val Ile Ser         #    60                                                                       - Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Va - #l Asp Ile Phe Ala Glu         #80                                                                           - Arg Leu Ser Ala Glu Lys Pro Gly Tyr Tyr Le - #u His Asn Gly Lys Trp         #                95                                                           - Val Lys Met Leu Ser Arg Glu Glu Thr Ile Th - #r Val Lys Asn Gly Gln         #           110                                                               - Ala Glu Thr Phe Thr Val Trp Arg Thr Val Hi - #s Gly Asn Ile Leu Gln         #       125                                                                   - Thr Asp Gln Thr Thr Gln Thr Ala Tyr Ala Ly - #s Ser Arg Ala Trp Asp         #   140                                                                       - Gly Lys Glu Val Ala Ser Leu Leu Ala Trp Th - #r His Gln Met Lys Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Asn Trp Gln Glu Trp Thr Gln Gln Ala Al - #a Lys Gln Ala Leu Thr         #               175                                                           - Ile Asn Trp Tyr Tyr Ala Asp Val Asn Gly As - #n Ile Gly Tyr Val His         #           190                                                               - Thr Gly Ala Tyr Pro Asp Arg Gln Ser Gly Hi - #s Asp Pro Arg Leu Pro         #       205                                                                   - Val Pro Gly Thr Gly Lys Trp Asp Trp Lys Gl - #y Leu Leu Pro Phe Glu         #   220                                                                       - Met Asn Pro Lys Val Tyr Asn Pro Gln Ser Gl - #y Tyr Ile Ala Asn Trp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Asn Ser Pro Gln Lys Asp Tyr Pro Ala Se - #r Asp Leu Phe Ala Phe         #               255                                                           - Leu Trp Gly Gly Ala Asp Arg Val Thr Glu Il - #e Asp Arg Leu Leu Glu         #           270                                                               - Gln Lys Pro Arg Leu Thr Ala Asp Gln Ala Tr - #p Asp Val Ile Arg Gln         #       285                                                                   - Thr Ser Arg Gln Asp Leu Asn Leu Arg Leu Ph - #e Leu Pro Thr Leu Gln         #   300                                                                       - Ala Ala Thr Ser Gly Leu Thr Gln Ser Pro Pr - #o Arg Arg Gln Leu Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Thr Leu Thr Arg Trp Asp Gly Ile Asn Le - #u Leu Asn Asp Asp Gly         #               335                                                           - Lys Thr Trp Gln Gln Pro Gly Ser Ala Ile Le - #u Asn Val Trp Leu Thr         #           350                                                               - Ser Met Leu Lys Arg Thr Val Val Ala Ala Va - #l Pro Met Pro Phe Asp         #       365                                                                   - Lys Trp Tyr Ser Ala Ser Gly Tyr Glu Thr Th - #r Gln Asp Gly Pro Thr         #   380                                                                       - Gly Ser Leu Asn Ile Ser Val Gly Ala Lys Il - #e Leu Tyr Glu Ala Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gln Gly Asp Lys Ser Pro Ile Pro Gln Ala Va - #l Asp Leu Phe Ala Gly         #               415                                                           - Lys Pro Gln Gln Glu Val Val Leu Ala Ala Le - #u Glu Asp Thr Trp Glu         #           430                                                               - Thr Leu Ser Lys Arg Tyr Gly Asn Asn Val Se - #r Asn Trp Lys Thr Pro         #       445                                                                   - Ala Met Ala Leu Thr Phe Arg Ala Asn Asn Ph - #e Phe Gly Val Pro Gln         #   460                                                                       - Ala Ala Ala Glu Glu Thr Arg His Gln Ala Gl - #u Tyr Gln Asn Arg Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Thr Glu Asn Asp Met Ile Val Phe Ser Pro Th - #r Thr Ser Asp Arg Pro         #               495                                                           - Val Leu Ala Trp Asp Val Val Ala Pro Gly Gl - #n Ser Gly Phe Ile Ala         #           510                                                               - Pro Asp Gly Thr Val Asp Lys His Tyr Glu As - #p Gln Leu Lys Met Tyr         #       525                                                                   - Glu Asn Phe Gly Arg Lys Ser Leu Trp Leu Th - #r Lys Gln Asp Val Glu         #   540                                                                       - Ala His Lys Glu Ser Gln Glu Val Leu His Va - #l Gln Arg                     545                 5 - #50                 5 - #55                           - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 555 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - Ser Asn Met Trp Val Ile Gly Lys Asn Lys Al - #a Gln Asp Ala Lys Ala         #                15                                                           - Ile Met Val Asn Gly Pro Gln Phe Gly Trp Ty - #r Ala Pro Ala Tyr Thr         #            30                                                               - Tyr Gly Ile Gly Leu His Gly Ala Gly Tyr As - #p Val Thr Gly Asn Thr         #        45                                                                   - Pro Phe Ala Tyr Pro Gly Leu Val Phe Gly Hi - #s Asn Gly Thr Ile Ser         #    60                                                                       - Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Va - #l Asp Ile Phe Ala Glu         #80                                                                           - Lys Leu Ser Ala Glu Lys Pro Gly Tyr Tyr Gl - #n His Asn Gly Glu Trp         #                95                                                           - Val Lys Met Leu Ser Arg Lys Glu Thr Ile Al - #a Val Lys Asp Gly Gln         #           110                                                               - Pro Glu Thr Phe Thr Val Trp Arg Thr Leu As - #p Gly Asn Val Ile Lys         #       125                                                                   - Thr Asp Thr Arg Thr Gln Thr Ala Tyr Ala Ly - #s Ala Arg Ala Trp Ala         #   140                                                                       - Gly Lys Glu Val Ala Ala Leu Leu Ala Trp Th - #r His Gln Met Lys Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Asn Trp Pro Glu Trp Thr Gln Gln Ala Al - #a Lys Gln Ala Leu Thr         #               175                                                           - Ile Asn Trp Tyr Tyr Ala Asp Val Asn Gly As - #n Ile Gly Tyr Val His         #           190                                                               - Thr Gly Ala Tyr Pro Asp Arg Gln Pro Gly Hi - #s Asp Pro Arg Leu Pro         #       205                                                                   - Val Pro Asp Gly Lys Trp Asp Trp Lys Gly Le - #u Leu Ser Phe Asp Leu         #   220                                                                       - Asn Pro Lys Val Tyr Asn Pro Gln Ser Gly Ty - #r Ile Ala Asn Trp Asn         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Ser Pro Gln Lys Asp Tyr Pro Ala Ser As - #p Leu Phe Ala Phe Leu         #               255                                                           - Trp Gly Gly Ala Asp Arg Val Thr Glu Ile As - #p Thr Ile Leu Asp Lys         #           270                                                               - Gln Pro Arg Phe Thr Ala Asp Gln Ala Trp As - #p Val Ile Arg Gln Thr         #       285                                                                   - Ser Leu Arg Asp Leu Leu Arg Leu Phe Leu Pr - #o Ala Leu Lys Asp Ala         #   300                                                                       - Thr Ala Asn Leu Ala Glu Asn Asp Pro Arg Ar - #g Gln Leu Val Asp Lys         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Ala Ser Trp Asp Gly Glu Asn Leu Val As - #n Asp Asp Gly Lys Thr         #               335                                                           - Tyr Gln Gln Pro Gly Ser Ala Ile Leu Asn Al - #a Trp Leu Thr Ser Met         #           350                                                               - Leu Lys Arg Thr Leu Val Ala Ala Val Pro Al - #a Pro Phe Gly Lys Trp         #       365                                                                   - Tyr Ser Ala Ser Gly Tyr Glu Thr Thr Gln As - #p Gly Pro Thr Gly Ser         #   380                                                                       - Leu Asn Ile Ser Val Gly Ala Lys Ile Leu Ty - #r Glu Ala Leu Gln Gly         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Lys Ser Pro Ile Pro Gln Ala Val Asp Le - #u Phe Gly Gly Lys Pro         #               415                                                           - Glu Gln Glu Val Ile Leu Ala Ala Leu Asp As - #p Ala Trp Gln Thr Leu         #           430                                                               - Ser Lys Arg Tyr Gly Asn Asp Val Thr Gly Tr - #p Lys Thr Pro Ala Met         #       445                                                                   - Ala Leu Thr Phe Arg Ala Asn Asn Phe Phe Gl - #y Val Pro Gln Ala Ala         #   460                                                                       - Ala Lys Glu Ala Arg His Gln Ala Glu Tyr Gl - #n Asn Arg Gly Thr Glu         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Asn Asp Met Ile Val Phe Ser Pro Thr Ser Gl - #y Asn Arg Pro Val Leu         #               495                                                           - Ala Trp Asp Val Val Ala Pro Gly Gln Ser Gl - #y Phe Ile Ala Pro Asp         #           510                                                               - Gly Lys Ala Asp Lys His Tyr Asp Asp Gln Le - #u Lys Met Tyr Glu Ser         #       525                                                                   - Phe Gly Arg Lys Ser Leu Trp Leu Thr Pro Gl - #n Asp Val Asp Glu His         #   540                                                                       - Lys Glu Ser Gln Glu Val Leu Gln Val Gln Ar - #g                             545                 5 - #50                 5 - #55                           - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 528 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - Ser Asn Ala Ala Ile Val Gly Ser Glu Lys Se - #r Ala Thr Gly Asn Ala         #                15                                                           - Leu Leu Phe Ser Gly Pro Gln Val Gly Phe Va - #l Ala Pro Gly Phe Leu         #            30                                                               - Tyr Glu Val Gly Leu His Ala Pro Gly Phe As - #p Met Glu Gly Ser Gly         #        45                                                                   - Phe Ile Gly Tyr Pro Phe Ile Met Phe Gly Al - #a Asn Asn His Phe Ala         #    60                                                                       - Leu Ser Ala Thr Ala Gly Tyr Gly Asn Val Th - #r Asp Ile Phe Glu Glu         #80                                                                           - Lys Leu Asn Thr Lys Asn Ser Ser Gln Tyr Le - #u Tyr Lys Gly Lys Trp         #                95                                                           - Arg Asp Met Glu Lys Arg Lys Glu Ser Phe Th - #r Val Lys Gly Asp Asn         #           110                                                               - Gly Glu Lys Lys Thr Val Glu Lys Ile Tyr Ty - #r Arg Thr Val His Gly         #       125                                                                   - Pro Val Ile Ser Arg Asp Glu Thr Asn Lys Va - #l Ala Tyr Ser Lys Tyr         #   140                                                                       - Val Ser Phe Arg Gly Thr Glu Glu Ala Gln Se - #r Met Ser Ala Tyr Met         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Ala Asn Trp Ala Lys Asn Leu Lys Glu Ph - #e Glu Asn Ala Ala Ser         #               175                                                           - Glu Tyr Thr Met Ser Leu Asn Trp Tyr Tyr Al - #a Asp Lys Lys Gly Asp         #           190                                                               - Ile Ala Tyr Tyr His Val Gly Arg Tyr Pro Va - #l Arg Asn Asn Lys Ile         #       205                                                                   - Asp Glu Arg Ile Pro Thr Pro Gly Thr Gly Gl - #u Tyr Glu Trp Lys Gly         #   220                                                                       - Phe Ile Pro Phe Lys Glu Asn Pro His Val Il - #e Asn Pro Lys Asn Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Tyr Val Val Asn Trp Asn Asn Lys Pro Ser Ly - #s Glu Trp Val Asn Gly         #               255                                                           - Glu Tyr Ser Tyr Tyr Trp Gly Glu Asp Asn Ar - #g Val Gln Gln Tyr Ile         #           270                                                               - Asn Gly Gly Met Glu Ala Arg Gly Lys Val Th - #r Leu Glu Asp Ile Asn         #       285                                                                   - Glu Ile Asn Tyr Thr Ala Ser Phe Ala Gln Le - #u Arg Ala Asn Leu Phe         #   300                                                                       - Lys Pro Leu Leu Ile Asp Val Leu Asp Lys As - #n Lys Ser Thr Asn Gly         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Tyr Thr Tyr Leu Ile Glu Lys Leu Glu Gl - #u Trp Asn Asn Leu Lys         #               335                                                           - Glu Asp Glu Asn Lys Asp Gly Tyr Tyr Asp Al - #a Gly Ile Ala Ala Phe         #           350                                                               - Phe Asp Glu Trp Trp Asn Asn Leu His Asp Ly - #s Leu Phe Met Asp Glu         #       365                                                                   - Leu Gly Asp Phe Tyr Gly Ile Thr Lys Glu Il - #e Thr Asp His Arg Tyr         #   380                                                                       - Gly Ala Ser Leu Ala Tyr Lys Asn Ile Ser Ly - #s Glu Ser Thr Asn Tyr         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Lys Trp Val Lys Trp Val Asn Val Asp Gln Gl - #u Lys Ile Ile Met Glu         #               415                                                           - Ser Thr Asn Glu Val Leu Ala Lys Leu Gln Se - #r Glu Lys Gly Leu Lys         #           430                                                               - Ala Glu Lys Trp Arg Met Pro Ile Lys Thr Me - #t Thr Phe Gly Glu Lys         #       445                                                                   - Ser Leu Ile Gly Ile Pro His Gly Tyr Gly Se - #r Met Thr Pro Ile Ile         #   460                                                                       - Glu Met Asn Arg Gly Ser Glu Asn His Tyr Il - #e Glu Met Thr Pro Lys         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Gly Pro Ser Gly Phe Asn Ile Thr Pro Pro Gl - #y Gln Ile Gly Phe Val         #               495                                                           - Lys Lys Asp Gly Thr Ile Ser Asp His Tyr As - #p Asp Gln Leu Val Met         #           510                                                               - Phe Ala Glu Trp Lys Phe Lys Pro Tyr Leu Ph - #e Asn Lys Lys Asp Ile         #       525                                                                   - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 553 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - Ser Asn Val Trp Leu Val Gly Lys Thr Lys Al - #a Ser Gly Ala Lys Ala         #                15                                                           - Ile Leu Leu Asn Gly Pro Gln Phe Gly Trp Ph - #e Asn Pro Ala Tyr Thr         #            30                                                               - Tyr Gly Ile Gly Leu His Gly Ala Gly Phe As - #n Ile Val Gly Asn Thr         #        45                                                                   - Pro Phe Ala Tyr Pro Ala Ile Leu Phe Gly Hi - #s Asn Gly His Val Ser         #    60                                                                       - Trp Gly Ser Thr Ala Gly Phe Gly Asp Gly Va - #l Asp Ile Phe Ala Glu         #80                                                                           - Gln Val Ser Pro Glu Asp Pro Asn Ser Tyr Le - #u His Gln Gly Gln Trp         #                95                                                           - Lys Lys Met Leu Ser Arg Gln Glu Thr Leu As - #n Val Lys Gly Glu Gln         #           110                                                               - Pro Ile Thr Phe Glu Ile Tyr Arg Thr Val Hi - #s Gly Asn Val Val Lys         #       125                                                                   - Arg Asp Lys Thr Thr His Thr Ala Tyr Ser Ly - #s Ala Arg Ala Trp Asp         #   140                                                                       - Gly Lys Glu Leu Thr Ser Leu Met Ala Trp Va - #l Lys Gln Gly Gln Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Asn Trp Gln Gln Trp Leu Asp Gln Ala Gl - #n Asn Gln Ala Leu Thr         #               175                                                           - Ile Asn Trp Tyr Tyr Ala Asp Lys Asp Gly As - #n Ile Gly Tyr Val His         #           190                                                               - Thr Gly His Tyr Pro Asp Arg Gln Ile Asn Hi - #s Asp Pro Arg Leu Pro         #       205                                                                   - Val Ser Gly Thr Gly Glu Trp Asp Trp Lys Gl - #y Ile Gln Pro Phe Ala         #   220                                                                       - Asn Asn Pro Lys Val Tyr Asn Pro Lys Ser Gl - #y Tyr Ile Ala Asn Trp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Asn Ser Pro Ala Lys Asn Tyr Pro Ala Se - #r Asp Leu Phe Ala Phe         #               255                                                           - Leu Trp Gly Ser Ala Asp Arg Val Lys Glu Il - #e Asp Asn Arg Ile Glu         #           270                                                               - Ala Tyr Asp Lys Leu Thr Ala Asp Asp Met Tr - #p Ala Ile Leu Gln Gln         #       285                                                                   - Thr Ser Arg Val Asp Leu Asn His Arg Leu Ph - #e Thr Pro Phe Leu Thr         #   300                                                                       - Gln Ala Thr Gln Gly Leu Pro Ser Asn Asp As - #n Ser Val Lys Leu Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Met Leu Gln Gln Trp Asp Gly Ile Asn Gl - #n Leu Ser Ser Asp Gly         #               335                                                           - Lys His Tyr Ile His Pro Gly Ser Ala Tyr Le - #u Asp Ile Trp Leu Lys         #           350                                                               - Glu Met Leu Lys Ala Thr Leu Gly Gln Thr Va - #l Pro Ala Pro Phe Asp         #       365                                                                   - Lys Trp Tyr Leu Ala Ser Gly Tyr Glu Thr Th - #r Gln Glu Gly Pro Thr         #   380                                                                       - Gly Ser Leu Asn Ile Ser Thr Gly Ala Lys Le - #u Leu Tyr Glu Ser Leu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Leu Glu Asp Lys Ser Pro Ile Ser Gln Ser Il - #e Asp Leu Phe Ser Gly         #               415                                                           - Gln Pro Gln Asn Asp Val Ile Arg Lys Thr Le - #u Asn Thr Thr Tyr Gln         #           430                                                               - Lys Met Ile Glu Lys Tyr Gly Asp Asn Pro Al - #a Asn Trp Gln Thr Pro         #       445                                                                   - Ala Thr Ala Leu Thr Phe Arg Glu Asn Asn Ph - #e Phe Gly Ile Pro Gln         #   460                                                                       - Ala Leu Pro Gln Glu Asn Phe His Gln Asn Gl - #u Tyr His Asn Arg Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Thr Glu Asn Asp Leu Ile Val Phe Thr Glu Gl - #u Gly Val Ser Ala Trp         #               495                                                           - Asp Val Val Ala Pro Gly Gln Ser Gly Phe Il - #e Ser Pro Gln Gly Lys         #           510                                                               - Pro Ser Pro His Tyr Gln Asp Gln Leu Ser Le - #u Tyr Gln Gln Phe Gly         #       525                                                                   - Lys Lys Pro Leu Trp Leu Asn Ser Glu Asp Va - #l Ala Pro Tyr Ile Glu         #   540                                                                       - Ser Thr Glu Thr Leu Ile Ile Glu Arg                                         545                 5 - #50                                                   __________________________________________________________________________

We claim:
 1. An isolated mutant prokaryotic Penicillin G acylase or itspreenzmye or preproenzyme having:an amino acid substitution at one ormore of the positions corresponding to A139 to A142 and A148 to A152 asset forth in SEQ ID NO: 27, B20 to B27, B31, B49 to B52, B56, B57, B65,B67 to B72, B154 to B157, B173 to B179, B239 to B241, B250 to B263, B379to B387, B390, B455, B474 to B480 as set forth in SEQ ID NO:32 inAlcaligenes faecalis Penicillin G acylase or its pre- or preproenzyme,wherein the substitution at position A141 results in a substituent aminoacid selected from the group consisting of Ala, Arg, Asn, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; andan altered substrate specificity or altered specific activity relativeto the corresponding wild-type unsubstituted Penicillin G acylase.
 2. Amutant acylase according to claim 1, wherein said acylase is obtainedfrom a member of the group consisting of Escherichia coli, Kluyveracitrophila, Providencia rettgeri, Arthrobacter viscosis, and Alcaligenesfaecalis.
 3. A mutant acylase according to claim 1, wherein the aminoacid substitution is one of the following:A141 (Gly) as set forth in SEQID NO: 27 to Cys, Thr, Met, Ala, Val, or Leu.
 4. A mutant acylaseaccording to claim 1, wherein the amino acid substitution is one of thefollowing:B24 (Phe) as set forth in SEQ ID NO: 32 to Ala, Leu or anyother amino acid.
 5. An isolated mutant prokaryotic Penicillin G acylaseor its preenzyme or preproenzyme having an amino acid substitution at aposition corresponding to A141 (Gly) as set forth in SEQ ID NO: 27wherein A141 (Gly) is changed to Cys, Thr, Met, Ala, Val or Leu.
 6. Anisolated mutant prokaryotic Penicillin G acylase or its preenzyme orpreproenzyme having an amino acid substitution at a positioncorresponding to B24 (Phe) as set forth in SEQ ID NO: 32 wherein B24(Phe) is changed to Ala or Leu.
 7. A nucleic acid sequence encoding amutant acylase as defined in claim
 1. 8. An expression vector comprisinga nucleic acid sequence as defined in claim 7 operably linked to apromoter sequence capable of directing its expression in a host cell. 9.A microorganism transformed with an expression vector as defined inclaim
 8. 10. A mic roorganism according to claim 9, which is amicroorganism of the genus Cephalosoprium or the genus Penicillium. 11.A process of preparing an isolated mutant acylase as defined in claim 1,which process comprises:culturing a microorganism as defined in claim 9,whereby said mutant acylase is produced; and isolating said acylase. 12.A method for deacylating a 6-acylated penicillanic acid, a 7-acylated(desacetoxy) cephalosporanic acid or a salt or ester thereof to form thecorresponding 6-amino penicillanic acid or 7-amino (desacetoxy)cephalosporanic acid or salt or ester thereof, respectively, whichcomprises contacting said 6-acylated or 7-acylated compound with amutant acylase as defined in claim 1 under conditions suitable fordeacylation to occur.
 13. A method for producing a semi-synthetic6-acylated penicillanic acid, a 7-acylated (desacetoxy) cephalosporanicacid or a salt or ester thereof which comprises contacting acorresponding 6-amino or 7-amino β-lactam or salt or ester thereof,respectively, and an acylating agent with a mutant acylase as defined inclaim 1 under conditions suitable for acylation to occur.